U.S. patent application number 13/000802 was filed with the patent office on 2012-05-24 for compositions and methods for promoting vascular barrier function and treating pulonary fibrosis.
Invention is credited to Dean Li, Nyall London, Weiquan Zhu.
Application Number | 20120129757 13/000802 |
Document ID | / |
Family ID | 42243345 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120129757 |
Kind Code |
A1 |
Li; Dean ; et al. |
May 24, 2012 |
COMPOSITIONS AND METHODS FOR PROMOTING VASCULAR BARRIER FUNCTION
AND TREATING PULONARY FIBROSIS
Abstract
Active agents and compositions that promote barrier function or
that inhibit permeability of the vascular endothelium associated
with pulmonary inflammation are described.
Inventors: |
Li; Dean; (Salt Lake City,
UT) ; London; Nyall; (Bountiful, UT) ; Zhu;
Weiquan; (Salt Lake City, UT) |
Family ID: |
42243345 |
Appl. No.: |
13/000802 |
Filed: |
December 11, 2009 |
PCT Filed: |
December 11, 2009 |
PCT NO: |
PCT/US2009/067746 |
371 Date: |
January 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61122265 |
Dec 12, 2008 |
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Current U.S.
Class: |
514/1.1 ;
514/384 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
9/14 20180101; A61K 38/1709 20130101; A61P 43/00 20180101; A61P
11/00 20180101; A61P 31/04 20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/1.1 ;
514/384 |
International
Class: |
A61K 31/4196 20060101
A61K031/4196; A61P 9/00 20060101 A61P009/00; A61K 38/17 20060101
A61K038/17 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with Government support under Grant
R01 HL077671 awarded by the National Institutes of Health. The
Government has certain rights to this invention.
Claims
1. A method of promoting vascular barrier function in a subject,
the method comprising: administering to the subject a
therapeutically effective amount of at least one Slit polypeptide,
wherein administering the at least one Slit polypeptide results in
the promotion of endothelial barrier function.
2. The method of claim 1, wherein the at least one Slit polypeptide
is a ligand of Robo4.
3. The method of claim 1, wherein the at least one Slit polypeptide
is at least one Slit2 polypeptide.
4. The method of claim 3, wherein the at least one Slit2
polypeptide is Slit2N (SEQ ID NO: 4).
5. The method of claim 1, wherein the at least one Slit polypeptide
comprises the polypeptide sequence of at least one of SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, and combinations, derivatives, homologs and
analogs thereof.
6. The method of claim 1, wherein the promotion of vascular barrier
function occurs in the presence of at least one mediator of
inflammation selected from the group consisting of a
lipopolysaccharide, TNF-.alpha., IL-1.beta., and combinations
thereof.
7. The method of claim 1, wherein the promotion of endothelial
barrier function comprises at least one of promoting the presence
of vascular endothelial cadherin (VE-cadherin) at the surface of
vascular endothelial cells and promoting the expression of
p120-catenin at the surface of vascular endothelial cells.
8. A method of promoting vascular barrier function in a subject,
the method comprising: administering to the subject a
therapeutically effective amount of at least one inhibitor of at
least one ARF GTP exchange factor (ARF-GEF), wherein modulating the
at least one ARF-GEF results in the inhibition of vascular
permeability in the subject.
9. The method of claim 8, wherein inhibiting the at least one
ARF-GEF results in the inhibition of at least one ADP ribosylation
factor (ARF).
10. The method of claim 9, wherein the at least one ARF is selected
from the group consisting of ARF6, ARF1, and combinations
thereof.
11. The method of any of claim 8, wherein the at least one
inhibitor is a small molecule compound that inhibits at least one
of the availability of the at least one ARF-GEF, the activation of
the at least one ARF-GEF, and the activity of the at least one
ARF-GEF.
12. The method of any of claim 8, wherein the at least one
inhibitor of at least one ARF-GEF comprises SecinH3.
13. The method of any of claim 8, wherein the at least one
inhibitor of at least one ARF-GEF comprises at least one of a
compound according to Formula 1 and a compound according to Formula
2.
14. The method of any of claim 8, wherein the at least one
inhibitor of at least one ARF-GEF is selected from one of:
##STR00004## and pharmaceutically acceptable salts, solvates or
hydrates thereof.
15. The method of claim 8, wherein the at least one inhibitor of at
least one ARF-GEF inhibits a cytohesin selected from the ARNO
family of cytohesins.
16. The method of claim 15, wherein the cytohesin is ARNO.
17. The method of claim 8, wherein the inhibition of vascular
permeability in the subject occurs in the presence of at least one
mediator of inflammation selected from the group consisting of a
lipopolysaccharide, TNF-.alpha., IL-1.beta., and combinations
thereof.
18. The method of claim 8, wherein the inhibition of vascular
permeability in the subject comprises at least one of promoting the
presence of VE-cadherin at the surface of vascular endothelial
cells and promoting the expression of p120-catenin at the surface
of vascular endothelial cells.
19. A method of promoting the presence in a subject of VE-cadherin
at the surface of vascular endothelial cells, the method
comprising: administering to the subject a therapeutic amount of at
least one Slit polypeptide; wherein administering to the subject at
least one of a Slit polypeptide promotes the presence in the
subject of VE-cadherin at the surface of vascular endothelial
cells.
20. The method of claim 19, wherein the at least one Slit
polypeptide is selected from at least one of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, and combinations, derivatives, homologs and analogs
thereof.
21. The method of claim 19, wherein promoting the presence in the
subject of VE-cadherin at the surface of vascular endothelial cells
occurs in the presence of at least one mediator of inflammation
selected from the group consisting of a lipopolysaccharide,
TNF-.alpha., IL-1.beta., and combinations thereof.
22. The method of claim 19, wherein administering the at least one
Slit polypeptide promotes expression of p120-catenin at the surface
of vascular endothelial cells.
23. A method of treating a subject with pulmonary vascular
inflammation, the method comprising: administering to the subject a
therapeutically effective amount of at least one Slit polypeptide,
wherein administering the at least one Slit polypeptide results in
the reduction of pulmonary vascular inflammation in the
subject.
24. The method of claim 23, wherein the at least one Slit
polypeptide is at least one Slit2 polypeptide.
25. The method of claim 24, wherein the at least one Slit2
polypeptide is Slit2N (SEQ ID NO: 4).
26. The method of claim 23, wherein the at least one Slit
polypeptide comprises the polypeptide sequence of at least one of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and combinations,
derivatives, homologs and analogs thereof.
27. The method of claim 23, wherein the reduction of vascular
inflammation occurs in the presence of at least one mediator of
inflammation selected from the group consisting of a
lipopolysaccharide, TNF-.alpha., IL-1.beta., and combinations
thereof.
28. The method of claim 23, wherein the administering the at least
one Slit polypeptide promotes the presence in the subject of
VE-cadherin at the surface of vascular endothelial cells.
29. The method of claim 23, wherein the administering the at least
one Slit polypeptide promotes the expression in the subject of
p120-catenin at the surface of vascular endothelial cells.
30. The method of claim 23, wherein treating vascular inflammation
comprises treating at least one of further comprising promoting in
the subject the expression of p120-catenin at the surface of
vascular endothelial cells.
31. A method of reducing vascular permeability associated with any
of acute pulmonary vascular edema, chronic pulmonary vascular
edema, acute pulmonary vascular inflammation, chronic pulmonary
vascular inflammation, pulmonary fibrosis, including idiopathic
pulmonary fibrosis, bacterial sepsis, or influenza infection, the
method comprising: administering to the subject a therapeutically
effective amount of at least one Slit polypeptide, wherein
administering the at least one Slit polypeptide reduces vascular
permeability associated with a pathological condition in the
subject.
32. The method of claim 31, wherein the pathological condition is
pulmonary fibrosis, including idiopathic pulmonary fibrosis.
33. The method of claim 31, wherein the at least one Slit
polypeptide is administered to the subject in the presence of at
least one mediator of inflammation selected from the group
consisting of a lipopolysaccharide, TNF-.alpha., IL-1.beta., and
combinations thereof.
34. The method of claim 31, wherein the administering at least one
Slit polypeptide promotes the presence of VE-cadherin at the
surface of vascular endothelial cells.
35. A method of treating a subject suffering from pulmonary
fibrosis, the method comprising: administering to the subject a
therapeutically effective amount of a compound selected from a
compounds according to Formula 1 and a compound according to
Formula 2.
36. The method of claim 35, comprising administering to the subject
a therapeutically effective amount of SecinH3 and pharmaceutically
acceptable salts, solvates or hydrates thereof.
37. The method of claim 35, comprising administering to the subject
a therapeutically effective amount of a compound selected from one
of: ##STR00005## and pharmaceutically acceptable salts, solvates or
hydrates thereof.
38. A method of inhibiting the occurrence of pulmonary fibrosis in
a subject, the method comprising: administering to the subject a
therapeutically effective amount of a compound selected from a
compounds according to Formula 1 and a compound according to
Formula 2.
39. The method of claim 38, comprising administering to the subject
a therapeutically effective amount of SecinH3 and pharmaceutically
acceptable salts, solvates or hydrates thereof.
40. The method of claim 38, comprising administering to the subject
a therapeutically effective amount of a compound selected from one
of: ##STR00006## and pharmaceutically acceptable salts, solvates or
hydrates thereof.
41. A method of treating a subject suffering from pulmonary
fibrosis, the method comprising: administering to the subject a
therapeutically effective amount of a Slit polypeptide.
42. The method of claim 41, comprising administering to the subject
a therapeutically effective amount of a Slit2 polypeptide.
43. The method of claim 42, wherein the Slit2 polypeptide is Slit2N
(SEQ ID NO: 4).
44. The method of claim 41, wherein the Slit polypeptide is
selected from one of the polypeptides represented by SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, and combinations, derivatives, homologs and
analogs thereof.
45. A method of inhibiting the occurrence of pulmonary fibrosis in
a subject, the method comprising: administering to the subject a
therapeutically effective amount of a Slit polypeptide.
46. The method of claim 45, comprising administering to the subject
a therapeutically effective amount of a Slit2 polypeptide.
47. The method of claim 45, wherein the Slit polypeptide is
selected from one of the polypeptides represented by SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, and combinations, derivatives, homologs and
analogs thereof.
Description
BACKGROUND
[0002] Acute and chronic pulmonary vascular inflammation and leak
are associated with multiple pathologic conditions. For example,
influenza infections and sepsis can be characterized by acute, and
potentially life-threatening, pulmonary vascular inflammation.
Additionally, chronic pulmonary vascular inflammation is associated
with the development and progression of pulmonary fibrosis.
Pulmonary fibrosis is the abnormal formation of fiber-like scar
tissue in the lungs, with the scar formation being preceded by, and
associated with, inflammation. Pulmonary fibrosis is a chronic
disease causing swelling and scarring of the alveoli and
interstitial tissues of the lungs. The cause of pulmonary fibrosis
is often never determined (i.e., idiopathic pulmonary fibrosis),
but in some instances, the development and progression pulmonary
fibrosis is associated with a disease or infection, such as, for
example, tuberculosis, systemic Lupus Erythematosis, systemic
sclerosis, one or more environmental conditions, such as, for
example, exposure to silica dust and asbestos, or even particular
drugs, such as nitiofurantoin, amiodarone, and bleomycin. Though
pulmonary fibrosis can be so mild as to cause few symptoms, it can
also be fatal.
SUMMARY OF THE INVENTION
[0003] Active agents and compositions that promote vascular barrier
function are described herein. Compositions described herein
include at least one active agent capable of promoting vascular
barrier function, and in one such embodiment, the compositions
described herein include an active agent that promotes the barrier
function of vascular endothelium. In another embodiment, the
compositions described herein include an active agent that promotes
vascular barrier function in endothelial tissue selected from one
of vascular endothelium of the lung, vascular endothelium of the
kidney and vascular endothelium of the spleen. In another
embodiment, a composition as described herein includes an active
agent that inhibits vascular permeability associated with pulmonary
inflammation, including vascular permeability associated with
conditions leading to or resulting from acute pulmonary
inflammation and chronic pulmonary inflammation. As illustrated by
the experimental examples provided herein, active agents according
to the present description, in particular embodiments, promote
vascular barrier function even in the presence of multiple
mediators of inflammation and vascular permeability, including, for
example, endotoxins (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta..
[0004] Methods for promoting vascular endothelial barrier function
are also provided herein. In one embodiment, a method for promoting
vascular endothelial barrier function includes treating one or more
vascular endothelial cells with an active agent as described
herein. In one such embodiment, the step of treating one or more
vascular endothelial cells may be carried out by administering to a
patient in need thereof a therapeutically effective amount of an
active agent as described herein. In particular embodiments,
treatment of the one or more vascular endothelial cells with the
active agent results in one or more of the following: preservation
of vascular endothelial barrier function; promotion of endothelial
barrier function in the presence of one or more mediators of
inflammation, including one or more of an endotoxin (e.g., LPS),
tumor necrosis factor (e.g., TNF-.alpha.), and IL-1.beta.;
inhibition of vascular leak in the presence of one or more
mediators of inflammation, including one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.; promotion of the presence of VE-cadherin at the surface
of vascular endothelial cells; and promotion of expression of
p120-catenin at the surface of vascular endothelial cell. In
another such embodiment, treatment of the one or more vascular
endothelial cells with the active agent restores, at least in part,
vascular barrier function after exposure of the vascular
endothelial cells to one or more mediators of inflammation, wherein
the one or more mediators of inflammation are selected from
including one or more of an endotoxin (e.g., LPS), tumor necrosis
factor (e.g., TNF-.alpha.), and IL-1.beta..
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1. Slit2N stabilizes the endothelium by enhancing
VE-cadherin localization at the cell surface. (A), In vitro
permeability was measured in HMVEC-lung stimulated with LPS,
TNF-.alpha., or IL-1.beta. in the presence of Mock or Slit2N. (B),
Robo4 or control siRNA knockdown HMVEC-lung were stimulated with
IL-1.beta. in the presence of Mock or Slit2N to assess permeability
in vitro. (C-E), HMVEC-lung were treated with Mock or Slit2N,
subjected to membrane fractionation and subsequent immunoblotting
for VE-cadherin (C), p120-catenin (D), or .beta.-catenin (E). (F),
HMVEC-lung were stimulated with Mock or Slit2N and subjected to
immunofluorescence for VE-cadherin (green). White arrows indicate
areas of enhanced VE-cadherin cell surface localization. For all
experiments N.gtoreq.3, * P<0.05, *** P<0.005, ****
P<0.001, errors bars represent s.e.m.
[0006] FIG. 2. Slit2N enhances a VE-cadherin/p120-catenin
interaction. (A), HMVEC-lung were stimulated with IL-1.beta. in the
presence of Mock or Slit2N and immunostained for VE-cadherin and
p120-catenin. White arrows indicate cell surface areas lacking
VE-cadherin or p120-catenin in Mock treated cells. Yellow arrows
indicate areas of enhanced cell surface localization of VE-cadherin
or p120-catenin in Slit2N treated cells. (B), HMVEC-lung were
stimulated with IL-1.beta. in the presence of Mock or Slit2N.
Lysates were subjected to immunoprecipitation for VE-cadherin
followed by immunoblot for p120-catenin and VE-cadherin. (C),
HMVEC-lung were stimulated with IL-1.beta. in the presence of Mock
or Slit2N. VE-cadherin internalization (green) was assessed and
areas of internalization are indicated by white arrows. (D), in
vitro permeability was measured in the presence of a control IgG or
VE-cadherin antibody. For all experiments N.gtoreq.3, * P<0.05,
** P<0.01, *** P<0.005, errors bars represent s.e.m.
[0007] FIG. 3. Slit2N inhibits LPS-induced permeability, protein
exudates, and cell infiltrates in vivo. (A), Robo4.sup.+/+ and
Robo4.sup.AP/AP mice were given an intravenous injection of Mock or
Slit2N followed by intratracheal instillation of 10 .mu.g LPS. Mice
later received an intravenous injection of Evans Blue Albumin (EBA)
and EBA accumulation in the lungs was used to assess vascular
permeability (N.gtoreq.4). (B-D), Alternatively, twenty four hours
after LPS administration, bronchoalveolar lavages were obtained and
assessed for protein content (B), total inflammatory cell
accumulation (C), or neutrophil accumulation (D) (N.gtoreq.5). e,
H&E staining was performed on lung sections from mice exposed
to LPS in the presence of Mock or Slit2N. (F), protein exudates
measured in mice treated with 3.3 .mu.g LPS(N=5) g-i, mice were
given an intravenous injection of Mock or Slit2N with control or
VE-cadherin blocking antibody followed by intratracheal
instillation of LPS. Bronchoalveolar lavages were obtained and
assessed for protein content (G), total inflammatory cell
accumulation (H), or neutrophil accumulation (N.gtoreq.5). *
P<0.05, *** P<0.005, **** P<0.001, error bars represent
s.e.m.
[0008] FIG. 4. Slit2N reduces permeability and mortality in a cecal
ligation and puncture model of sepsis. (A), Mice were subjected to
CLP or sham operation. Mice were given an intravenous injection of
Evans Blue Albumin (EBA) and EBA accumulation measured in the
kidney (A) or spleen (B) to assess vascular permeability (N=5).
(C), Robo4.sup.+/+ mice were subjected to CLP and treated with Mock
or Slit2N and survival assessed (Mock treated N=15, Slit2N treated
N=14). Cytokine (D) or chemokine (E) levels in the serum of Mock or
Slit2N treated CLP mice (N=6). (F), Robo4.sup.AP/AP mice were
subjected to CLP and treated with Mock or Slit2N and survival
assessed (Mock treated N=13, Slit2N treated N=13). * P<0.05, **
P<0.01, **** P<0.001, error bars represent s.e.m.
[0009] FIG. 5. Slit2N reduces mortality in models of H5N1
infection. (A), Balb/c mice were infected intranasally with H5N1
virus. Mice were given an intravenous injection of Evans Blue
Albumin (EBA) and EBA accumulation was measured in the lungs to
assess vascular permeability (N=5). (B), mouse survival after H5N1
infection (Mock treated N=20, Slit2N treated N=20). (C), H&E
staining was performed on lung sections from H5N1 infected mice 6
days after infection. White arrows in the upper left panel indicate
accumulation of edema fluid around a pulmonary arteriole. The upper
middle panel demonstrates exuberant alveolar inflammation. The
black arrow in the upper right panel indicates the presence of
foamy macrophages. (D), H5N1 viral titers were measured 6 days
post-infection (N=3 groups of pooled mice). Cytokine (E) or
chemokine (F) levels measured in lung homogenates 6 days
post-infection (N=3 groups of pooled mice).
[0010] FIG. 6. Slit reduces vascular leak caused by multiple
inflammatory stimuli through enhancing VE-cadherin at the cell
surface. (A), Under normal conditions, alveolar capillaries provide
a semi-permeable barrier. (B), Inflammatory stimuli cause a large
release of cytokines leading to internalization of VE-cadherin and
disruption of barrier function. This results in vascular leak and
accumulation of protein-rich edema fluid in the alveolar space.
(C), Slit enhances vascular barrier function against multiple
cytokines by enhancing VE-cadherin at the cell surface.
[0011] FIG. 7. Recombinant Slit peptides as small as Slit2-D1 (40
kD) are active. In FIG. 7A, different constructs for the Slit
protein are depicted. The four leucine rich domains (LRR), the
epidermal growth factor homology region (EGF) and the c-terminal
tags (MYC/HIS) are indicated. Inhibition of VEGF mediated
endothelial cell migration by the different Slit constructs (2 nM)
is shown in FIG. 7B.
[0012] FIG. 8. SecinH3 inhibits Bleomycin-induced fibrosis. 6-8
week old BL/6 mice were given an intranasal instillation of saline
or 0.05 U Bleomycin. 100 uL of Vehicle or 30 uM SecinH3 was
administered twice a day via intraperitoneal injection. Pulmonary
fibrosis was assessed by Sircol collagen assay. N.gtoreq.7 animals
per group, * P<0.05.
[0013] FIG. 9. The chemical structure of SecinH3.
[0014] FIG. 10. An illustration of the Robo4 signaling pathway.
[0015] FIG. 11. Robo4 expression is increased in the lung 6 hours
after LPS instillation.
[0016] FIG. 12. The effect of administering a Slit2 protein on the
survival of mice infected with Avian Flu Virus in accordance with a
mouse model of avian flu.
[0017] FIG. 13. (a) Slit2 significantly reduced Bleomycin-induced
EBA accumulation in the lung of Robo4.sup.+/+ mice, eleven days
after Bleomycin administration. The effect of Slit2 was lost in
Robo4.sup.AP/AP mice. (b) Slit2 also significantly reduced
Bleomycin-induced pulmonary fibrosis. This effect was lost in
Robo4.sup.AP/AP mice, indicating that Slit2 acted directly upon the
endothelium to reduce pulmonary fibrosis. (c) Histologic
examination of the lung using a trichrome stain to enhance the
visualization of collagen deposition confirmed the effect of Slit2
in a Robo4-dependent manner.
[0018] FIG. 14. Slit inhibits LPS-induced cell infiltrates in a
dose-dependent manner. (A) Robo4 or control siRNA knockdown
HMVEC-lung were assessed for Robo4 expression by immunoblot. (B)
Confocal images of the Z-axis are shown below and to the side as
indicated by the yellow lines. Enhanced junctional thickness is
observed in Slit2N treated cells. (C-D) Mice were subjected to
LPS-induced ALI in the presence of increasing levels of Slit2N.
Cell infiltrate and neutrophil are shown. N=4, *P<0.05.
[0019] FIG. 15. Slit2N reduces LPS-induced ALI as assessed by
quantitative histology. Robo4.sup.+/+ and Robo4.sup.AP/AP mice were
subjected to LPS-induced ALI in the presence of Mock of Slit2N.
Lung sections from these mice were stained for H&E and
quantitative histology to assess the degree of lung injury
performed by blinded investigators. N=3, *** P<0.005.
[0020] FIG. 16. Slit does not reduce migration of primary human
PMNs. (A) Cells were subjected to migration to the leukocyte
chemoattractant fMLP in the presence of Mock Slit2. (B) RNA was
isolated from hPMNs and subjected to quantitative PCR. Brain cDNA
was used as a positive control. N=3, ***P<0.005, error bars
represent s.e.m.
[0021] FIG. 17. Slit2 protein is expressed throughout the lung in
close proximity to the endothelium. Slit2 co-localizes with Robo4
alkaline phosphatase (AP) expression indicated by arrows.
[0022] FIG. 18. Significant lung injury is absent during CLP. (A-C)
Mice were subjected to CLP and treated with Mock or Slit2N. Lung
sections were stained with H&E (A) and quantitative histology
to assess the degree of lung injury performed (B) N=3. (C)
Permeability in the lungs of mice was assessed using Evans Blue
Albumin. N=5.
[0023] FIG. 19. Loss of Robo4 does not affect patterning of the
vascular endothelium in the early developing lung. A-C, D-F and G-I
show Robo4.sup.+/+, Robo4.sup.AP/AP and Robo4.sup.AP/AP.about.E12.5
lungs, respectively, stained for epithelium (E-cadherin) and
vasculature (CD31). Arrows indicate the distal extent of the left
pulmonary artery EC tube. B, E, and H are magnified views of the
distal branches of the first left lateral secondary airway branch
with the crossbar denoting the thickness of the CD31+ plexus
compartment extending linearly outward from the vertex of the
distal branches. C, F, and I show magnified view of the distal left
pulmonary artery EC tube regions stained with an antibody directed
against CD31. The locations of the first, second and third left
dorsal secondary airway buds are denoted by D1, D2 and D3.
[0024] FIG. 20. Loss of Robo4 does not affect patterning of the
vascular endothelium in the developing lung. A-B, C-D and E-F show
Robo4.sup.+/+, Robo4.sup.+/AP and Robo4.sup.AP/AP.about.E14.5
lungs, respectively, stained for lung epithelium (E-cadherin) and
vascular development (CD31). Arrows denote the left pulmonary
artery located lateral to the left primary bronchus. The dark
arrowheads in A, C, and E mark the branch of the right pulmonary
artery supplying the right accessory lobe and located posterior to
the right secondary airway branch to the accessory lobe.
DETAILED DESCRIPTION
I. Definitions
[0025] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed active agents,
compositions and methods. These and other materials are disclosed
herein, and it is understood that when combinations, subsets,
interactions, groups, etc. of these materials are disclosed that,
while specific reference of each various individual and collective
combinations and permutation of these compounds may not be
explicitly disclosed, each is specifically contemplated and
described herein. For example, if a polypeptide is disclosed and
discussed and a number of modifications that can be made to a
number of molecules including the polypeptide are discussed, each
and every combination and permutation of polypeptide and the
modifications that are possible are specifically contemplated,
unless specifically indicated to the contrary. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F, and an example of a combination molecule, A-D, is
disclosed, then even if each is not individually recited, each is
individually and collectively contemplated. Thus, is this example,
each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F
are specifically contemplated and should be considered disclosed
from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. Likewise, any subset or combination of these is
also specifically contemplated and disclosed. Thus, for example,
the sub-group of A-E, B-F, and C-E are specifically contemplated
and should be considered disclosed from disclosure of A, B, and C;
D, E, and F; and the example combination A-D. This concept applies
to all aspects of this application including, but not limited to,
steps in methods of making and using the disclosed compositions.
Thus, if there are a variety of additional steps that can be
performed it is understood that each of these additional steps can
be performed with any specific embodiment or combination of
embodiments of the disclosed methods, and that each such
combination is specifically contemplated and should be considered
disclosed.
[0026] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, equivalents
to the specific embodiments of the method and compositions
described herein. Such equivalents are intended to be encompassed
by the included claims.
[0027] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the inventions described
herein.
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the meanings that would be commonly understood by
one of skill in the art in the context of the present
specification.
[0029] It must be noted that as used herein and in the appended
claims, the singular forms a, an, and the include plural reference
unless the context clearly dictates otherwise. Thus, for example,
reference to a polypeptide includes a plurality of such
polypeptides, reference to the polypeptide is a reference to one or
more polypeptides and equivalents thereof known to those skilled in
the art, and so forth.
[0030] Optional or optionally means that the subsequently described
event, circumstance, or material may or may not occur or be
present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0031] Ranges can be expressed herein as from about one particular
value, and/or to about another particular value. When such a range
is expressed, another embodiment includes from the one particular
value and/or to the other particular value. Similarly, when values
are expressed as approximations, by use of the antecedent about, it
will be understood that the particular value forms another
embodiment. It will be further understood that the endpoints of
each of the ranges are significant both in relation to the other
endpoint, and independently of the other endpoint. It is also
understood that there are a number of values disclosed herein, and
that each value is also herein disclosed as about that particular
value in addition to the value itself. For example, if the value 10
is disclosed, then about 10 is also disclosed. It is also
understood that each unit between two particular units are also
disclosed. For example, if 10 and 15 are disclosed, then 11, 12,
13, and 14 are also disclosed.
[0032] Alkyl refers to an optionally substituted hydrocarbon group
joined by single carbon-carbon bonds and having 1 to 8 carbon atoms
joined together. The alkyl hydrocarbon group may be straight-chain
or contain one or more branches. These groups include methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl,
hexyl, and the like. Lower alkyl refers to optionally substituted
branched- or straight-chain alkyl having 1 to 4 carbons.
[0033] Alkenyl refers to an optionally substituted hydrocarbon
group containing at least one carbon-carbon double bond between the
carbon atoms and containing 2-8 carbon atoms joined together. The
alkenyl hydrocarbon group may be branched or straight-chain.
[0034] Cycloalkyl refers to an optionally substituted cyclic alkyl
or an optionally substituted non-aromatic cyclic alkenyl and
includes monocyclic and multiple fused ring structures such as
bicyclic and tricyclic. The cycloalkyl may have, for example, 3 to
15 carbon atoms. In one embodiment, cycloalkyl has 5 to 12 carbon
atoms. Examples of suitable cycloalkyl groups include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl and the like.
[0035] Heterocycle refers to optionally substituted saturated or
partially saturated non-aromatic ringed moieties including at least
one non-carbon atom. Heterocyclic moieties typically comprise a
single ring or multiple fused ring structures, such as bicyclic and
tricyclic. In one embodiment, the ring(s) is 5 to 6-membered and
typically contains 1 to 3 non-carbon atoms. Non-carbon atoms for
heterocycle may be independently selected from nitrogen, oxygen and
sulfur.
[0036] Aryl refers to an optionally substituted aromatic group with
at least one ring having a conjugated pi-electron ring system, and
includes monocyclic and multiple fused ring structures such as
bicyclic and tricyclic. Aryl includes optionally substituted
carbocyclic aryl. Examples of suitable aryl groups include phenyl,
naphthyl, anthracenyl, phenanthrenyl and the like.
[0037] Heterocyclic aryl refers to an optionally substituted
aromatic group with at least one ring having a conjugated
pi-electron ring system including at least one non-carbon atom.
Heterocyclic aryl moieties typically comprise one ring or multiple
fused ring structures, such as bicyclic and tricyclic. Examples of
suitable heterocyclic aryl groups include furanyl, thienyl,
pyrrolyl, imidazolyl, pyridinyl, and the like.
[0038] Alkoxy refers to oxygen joined to an alkyl group. Lower
alkoxy refers to oxygen joined to a lower alkyl group. In one
embodiment, the oxygen is joined to an unsubstituted alkyl 1 to 4
carbons in length. For example, the alkoxy may be methoxy, ethoxy
and the like.
[0039] Alkylene refers to an optionally substituted hydrocarbon
chain containing only carbon-carbon single bonds between the carbon
atoms. The alkylene chain has 1 to 6 carbons and is attached at two
locations to other functional groups or structural moieties.
Examples of suitable alkylene groups include methylene, ethylene
and the like.
[0040] As used herein, small molecule refers to low molecular
weight compounds. For example, in particular embodiments, such
small molecule compounds are compounds the exhibit a molecular
weight of between 50 daltons to 800 daltons. In alternative
embodiments, a small molecule as described herein exhibit a
molecular weight selected from the ranges of between 100 daltons
and 500 daltons and between 250 daltons to 475 daltons.
[0041] As used herein, the term subject means any target of
administration. The subject can be a vertebrate, for example, a
mammal. Thus, the subject can be a human. The term does not denote
a particular age or sex. Thus, adult and newborn subjects, as well
as fetuses, whether male or female, are intended to be covered. The
term patient refers to a subject afflicted with a pathologic
condition. The term patient includes human and veterinary
subjects.
[0042] Inhibit, inhibiting, and inhibition mean to prevent,
decrease, inactivate, or reverse an activity, response, condition,
disease, or other biological parameter. Inhibit, inhibiting, and
inhibition can include, but is not limited to the complete ablation
of the activity, response, condition, or disease. Inhibit,
inhibiting, and inhibition can also include, for example, a slowing
or reduction of an activity, response, condition, disease, or other
biological parameter as compared to a native level, with the term
native level referring to a level evident in the absence of an
inhibiting agent. Inhibit, inhibiting, and inhibition can also
include, for example, reversal of an activity, response, condition,
disease, or other biological parameter as compared to a native
level, with the term native level referring to a level evident in
the absence of an inhibiting agent. In this context, a reduction
can be any measurable reduction. In particular embodiments, a
reduction can be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, or any amount of reduction in between the
specifically recited percentages, as compared to a native
level.
[0043] Promote, promotion, and promoting refer to a preservation,
restoration, or increase in an activity, response, condition, or
other biological parameter. Promote, promotion, and promoting can
include but is not limited to the initiation of an activity,
response, condition, or biological parameter. Alternatively,
promote, promotion, and promoting can include preservation of an
activity, response, condition, or other biological parameter in
light of a condition that would otherwise degrade, reduce or
eliminate the relevant activity, response, condition, or other
biological parameter. Promote, promotion, and promoting can also
include, for example, an increase in the activity, response,
condition, or biological parameter as compared to a native or
control level. In particular embodiments, the increase in an
activity, response, condition, or other biological parameter can be
an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
or more, including any amount of increase in between the
specifically recited percentages, as compared to native or control
levels, with the term native level referring to a level evident in
the absence of an promoting agent.
[0044] The term carrier means a compound, composition, substance,
or structure that, when in combination with a compound or
composition, aids or facilitates preparation, storage,
administration, delivery, effectiveness, selectivity, or any other
feature of the compound or composition for its intended use or
purpose. For example, a carrier can be used in providing a
pharmaceutical formulation and can be selected to minimize any
degradation of the active agent and to minimize any adverse side
effects in the subject.
[0045] As used herein, the terms treat, treating, and treatment
refer to a therapeutic benefit, whereby the detrimental effect(s)
or progress of a particular pathologic condition, disease,
condition, event or injury is prevented, reduced, halted, reversed
or slowed.
[0046] A therapeutically effective amount is the amount of compound
which achieves a therapeutic benefit, such as, for example, by
inhibiting or reversing an activity, response, condition, disease,
or other parameter associated with a pathologic condition. A
therapeutically effective amount may be an amount which relieves,
at least to some extent, one or more symptoms of a pathologic
condition in a subject; returns to normal, either partially or
completely, one or more physiological or biochemical parameters
associated with or causative of a pathologic condition; and/or
reduces the likelihood of the onset of a pathologic condition.
[0047] The terms pathologic or pathologic conditions refer to any
deviation from a healthy, normal, or efficient condition which may
be the result of a disease, condition, event or injury.
[0048] As the terms are used herein, protein and peptide refer to
polypeptide molecules generally and are not used to refer to
polypeptide molecules of any specific size, length or molecular
weight. Protein variants and derivatives are well understood to
those of skill in the art and can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Immunogenic fusion protein derivatives, such as those described in
the examples, are made by fusing a polypeptide sufficiently large
to confer immunogenicity to the target sequence by cross-linking in
vitro or by recombinant cell culture transformed with DNA encoding
the fusion. Deletions are characterized by the removal of one or
more amino acid residues from the protein sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein molecule. These variants ordinarily are prepared
by site-specific mutagenesis of nucleotides in the DNA encoding the
protein, thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. Techniques for
making substitution mutations at predetermined sites in DNA having
a known sequence are well known, for example M13 primer mutagenesis
and PCR mutagenesis. Amino acid substitutions are typically of
single residues, but can occur at a number of different locations
at once; insertions usually will be on the order of about from 1 to
10 amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional variants are well understood in the art
and are those in which at least one residue has been removed and a
different residue inserted in its place. Typically, substitutions
made in the formation of substitutional variants are conservative
substitutions, as are well known in the art, and often
substitutional variants may be made to made to enhance one or more
characteristics of a polypeptide molecule, such as, for example,
circulating half-life, stability, etc., while retaining or
improving the biologic activity of polypeptide.
[0049] Substantial changes in function or immunological identity
are made by selecting substitutions differ in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the protein properties will be those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or
by) an electronegative residue, e.g., glutamyl or aspartyl; or (d)
a residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine,
in this case, (e) by increasing the number of sites for sulfation
and/or glycosylation.
[0050] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the polypeptides
provided herein.
[0051] Substitutional or deletional mutagenesis can be employed to
insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation
(Ser or Thr). Deletions of cysteine or other labile residues also
may be desirable. Deletions or substitutions of potential
proteolysis sites, e.g. Arg, is accomplished for example by
deleting one of the basic residues or substituting one by
glutaminyl or histidyl residues.
[0052] In the context of recombinantly produced polypeptides,
certain post-translational derivatizations are the result of the
action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the o-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco pp
79-86 [1983], incorporated herein by reference), acetylation of the
N-terminal amine and, in some instances, amidation of the
C-terminal carboxyl.
[0053] It is understood that one way to define the derivatives,
analogs and homologs of the polypeptides disclosed herein is
through defining the derivatives, analogs and homologs in terms of
homology/identity to specific known sequences. Those of skill in
the art readily understand how to determine the homology of two
proteins. For example, the homology can be calculated after
aligning the two sequences so that the homology is at its highest
level.
[0054] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0055] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
polypeptides. For example, there are numerous D amino acids or
amino acids which have a different functional substituent then the
amino acids shown in Table 1. The opposite stereo isomers of
naturally occurring peptides are disclosed, as well as the stereo
isomers of peptide analogs. These amino acids can readily be
incorporated into polypeptide chains by charging tRNA molecules
with the amino acid of choice and engineering genetic constructs
that utilize, for example, amber codons, to insert the analog amino
acid into a peptide chain in a site specific way (Thorson et al.,
Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in
Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic
Engineering Reviews 13:197-216 (1995), Cahill et al., TIBS,
14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba
and Hennecke, Bio/technology, 12:678-682 (1994) all of which are
herein incorporated by reference).
[0056] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference).
[0057] As used herein, vascular permeability refers to the capacity
of small molecules (e.g., ions, water, nutrients), large molecules
(e.g., proteins and nucleic acids) or even whole cells (lymphocytes
on their way to the site of inflammation) to pass through a blood
vessel wall.
II. Robo4 Signaling Pathway
[0058] A signaling pathway whereby Robo4 signaling inhibits
pathologic angiogenesis and neovascularization is described, in
International Publication No. WO 2009/129408, International
Publication No. WO 2008/073441, and Jones et al. (C. A. Jones et
al., 2008. Robo4 stabilizes the vascular network by inhibiting
pathologic angiogenesis and endothelial hyperpermeability. Nat Med
14:448-453). As is described in these references, expression of
Robo4 confers responsiveness to Slit2, and Slit2-Robo4 signaling
negatively regulates cellular protrusive activity stimulated by
cell adhesion. Such negative regulation is mediated by interaction
of Robo4 with the adaptor protein, paxillin, and its paralogues,
which recruits ARF-GAPs such as GIT1, leading to local inactivation
of ADP ribosylation factor 6 (ARF6). This signaling pathway
(illustrated in FIG. 10) thereby interferes with adhesion-mediated
Rac1 activation and cell protrusion. The signaling pathway
described in FIG. 10 presents multiple targets for modulating the
Robo-4 signaling pathway, and Jones et al., International
Publication No. WO 2009/129408, and International Publication No.
WO 2008/073441, further describe that modulation of ARF-GAPs and
ARF-GEFs involved in the Robo4 signaling pathway can be
accomplished without Slit/Robo4 signaling. The contents of each of
Jones et al., International Publication No. WO 2009/129408, and
International Publication No. WO 2008/073441 are incorporated
herein by this reference.
[0059] Robo4 signaling works to preserve vascular integrity in the
presence of multiple different mediators of inflammation. For
example, the Robo-4 signaling pathway can be utilized to preserve
vascular integrity in the presence of endotoxin (e.g.,
lipopolysaccharide or "LPS"), tumor necrosis factor (e.g.,
TNF-.alpha.), and interleukin-1.beta. ("IL-1.beta."), each of which
is a known mediators of inflammation (Dinarello, C. A. 1997.
Proinflammatory and anti-inflammatory cytokines as mediators in the
pathogenesis of septic shock. Chest 112:321 S-329S). Moreover, the
Robo-4 signaling pathway functions to preserve endothelial barrier
function in multiple different tissues, such as, for example, in
the lung, the kidney, and the spleen. Even further, the Robo4
signaling pathway serves not only to preserve vascular integrity in
conditions associated with an acute inflammatory response, but also
the Robo-4 signaling pathway can be utilized to preserve
endothelial barrier function in-vivo in models of acute and chronic
pulmonary inflammation.
II. Active Agents & Compositions
[0060] The active agents and compositions described herein serve to
promote vascular barrier function. In each embodiment, the
compositions described herein include at least one active agent
capable of promoting vascular barrier function, and in one such
embodiment, the compositions described herein include an active
agent that promotes the barrier function of vascular endothelium.
In another embodiment, the compositions described herein include an
active agent that promotes vascular barrier function in endothelial
tissue selected from one of vascular endothelium of the lung,
vascular endothelium of the kidney and vascular endothelium of the
spleen. In another embodiment, a composition as described herein
includes an active agent that inhibits vascular permeability
associated with conditions leading to acute pulmonary inflammation
as well as conditions associated with chronic pulmonary
inflammation, such as the development and progression of pulmonary
fibrosis. As illustrated by the experimental examples provided
herein, active agents according to the present description, in
particular embodiments, promote vascular barrier function even in
the presence of multiple mediators of inflammation and vascular
permeability, including, for example, endotoxins (e.g., LPS), tumor
necrosis factor (e.g., TNF-.alpha.), and IL-1.beta..
[0061] In specific embodiments, the active agents and compositions
described herein are suitable for treating a subject suffering from
a pathological condition such as pulmonary fibrosis, as well as
other conditions associated with acute pulmonary vascular
inflammation. In one such embodiment, a composition as described
herein includes an active agent that inhibits one or more of acute
pulmonary vascular edema, acute pulmonary vascular inflammation,
and chronic pulmonary vascular inflammation associated with
development or progression of pulmonary fibrosis, including
idiopathic pulmonary fibrosis. In another such embodiment, a
composition as described herein includes an active agent that
promotes vascular barrier function in animals, including humans,
exposed to a microbial endotoxin, or suffering from an influenza
infection, such as an avian flu infection. In still further
embodiments, compositions described herein include an active agent
that promotes vascular barrier function and inhibits vascular
permeability associated with pathological conditions such as
bacterial sepsis, or influenza infection, such as an avian flu
infection.
[0062] In the vascular endothelium, critical stabilizing
interactions are mediated by the adherens junction protein,
vascular endothelial cadherin (VE-cadherin) (Dejana, E., F.
Orsenigo, and M. G. Lampugnani. 2008. The role of adherens
junctions and VE-cadherin in the control of vascular permeability.
J Cell Sci 121:2115-2122; Vestweber, D. 2008. VE-cadherin: the
major endothelial adhesion molecule controlling cellular junctions
and blood vessel formation. Arterioscler Thromb Vasc Biol
28:223-232). VE-cadherin surface expression is regulated by the
association of p120-catenin with VE-cadherin, and the association
of p120-catenin with VE-cadherin is known to inhibit VE-cadherin
internalization from the cell surface and promote vascular
stability (Potter, M. D., S. Barbero, and D. A. Cheresh. 2005.
Tyrosine phosphorylation of VE-cadherin prevents binding of p120-
and beta-catenin and maintains the cellular mesenchymal state. J
Biol Chem 280:31906-31912; Xiao, K., J. Garner, K. M. Buckley, P.
A. Vincent, C. M. Chiasson, E. Dejana, V. Faundez, and A. P.
Kowalczyk. 2005. p120-Catenin regulates clathrin-dependent
endocytosis of VE-cadherin. Mol Biol Cell 16:5141-5151). In
particular embodiments, the active agents described herein promote
the presence of VE-cadherin at cell surface junctions. In one such
embodiment, an active agent according to the present description
promotes cell surface p120-catenin expression. In promoting
expression of cell surface p120-catenin, it is presently believed,
without being bound by a particular theory, that such embodiments
preserve the association between VE-cadherin and p120-catenin and,
thereby, promote vascular integrity by reducing VE-cadherin
endocytosis, such as may otherwise occur in the presence of one
more mediators of inflammation (e.g., one or more cytokines).
Therefore, in particular embodiments, the active agents described
herein promote vascular barrier function in animals, including
humans, by one or both of promoting the presence of VE-cadherin at
the surface of endothelial cells, such as vascular endothelial
cells, and promoting expression of p120-catenin at the surface of
endothelial cells, such as vascular endothelial cells
[0063] An active agent according to the present description can
include a Slit polypeptide, such as Slit2 polypeptide. When
discussing Slit2 polypeptides as contemplated herein, full-length
Slit2 proteins, as well as derivatives, analogs and homologs of
full-length Slit2 proteins are contemplated, provided that such
polypeptides promote endothelial barrier function in the presence
of one or more mediators of inflammation, including one or more of
an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta., inhibit vascular leak in the presence
of one or more mediators of inflammation, including one or more of
an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta., promote of the presence of
VE-cadherin at the surface of vascular endothelial cells, or
promote expression of p120-catenin at the surface of vascular
endothelial cells. A derivative polypeptide molecule refers to a
polypeptide formed from native compounds either directly or by
modification or partial substitution. A homolog polypeptide
molecule refers to a polypeptide product of a particular gene
derived from a different species. An analog polypeptide molecule is
a polypeptide that is similar in structure, but not identical, and
differs with respect to number or nature of amino acids included in
a referenced polypeptide sequence. For example, an analog to a
given polypeptide will exhibit a level of sequence homology, but
may include one or more amino acid substitutions or deletions.
[0064] In specific embodiments, where the active agent is a Slit2
polypeptide, the active agent may be selected from mammalian Slit2
polypeptides, such as a human Slit2 polypeptide. Where the active
agent is a mammalian Slit2, the active agent may be selected from
known, full-length, naturally occurring, mammalian Slit2
polypeptides, such as, for example, the Slit2 polypeptide
represented by SEQ ID NO: 1, as well as derivative, analogs and
homologs thereof, that are capable of on one or more of the
following: promoting endothelial barrier function in the presence
of one or more mediators of inflammation, including one or more of
an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibiting vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promoting the presence of VE-cadherin
at the surface of vascular endothelial cells; and promoting
expression of p120-catenin at the surface of vascular endothelial
cells. As will be readily appreciated, naturally occurring Slit2
polypeptides can be isolated and purified according to techniques
known in the art (Wang, K H et al. 1999. Biochemical purification
of a mammalian slit protein as a positive regulator of sensory axon
elongation and branching. Cell March 19; 96(6):771-84; Chedotal, A.
2007. Slits and their receptors. Adv Exp Med Biol 621:65-80).
[0065] In addition to naturally occurring Slit2 polypeptides, a
Slit2 active agent as contemplated herein may be obtained through
recombinant or synthetic production techniques well known in the
art. Even further, the active agent may be selected from
derivatives, analogs, or homologs of naturally occurring,
recombinant, or synthetic mammalian Slit2 polypeptides. In specific
embodiments, a Slit2 active agent can be selected from a fragment
of a naturally occurring Slit2 protein, such as the fragment
represented either of SEQ ID NO: 2 or SEQ ID NO: 3, as well as
derivatives, analogs and homologs thereof, capable of one or more
of the following: promoting endothelial barrier function in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibiting vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promoting the presence of VE-cadherin
at the surface of vascular endothelial cells; and promoting
expression of p120-catenin at the surface of vascular endothelial
cells.
[0066] In still other embodiments, the Slit2 polypeptides
represented by SEQ ID NO: 4 through SEQ ID NO: 12 may be used as an
active agent. In particular, an active agent according to the
present description may be selected from Slit2N (SEQ ID NO: 4), the
Slit2 polypeptide represented by SEQ ID NO: 5, Slit2.DELTA.P (SEQ
ID NO: 6), Slit2 D1 (SEQ ID NO: 7), Slit2 D1-D2 (SEQ ID NO: 8),
Slit2 D1-D3 (SEQ ID NO: 9), Slit2 D1-D4 (SEQ ID NO: 10), Slit2
D1-E5 (SEQ ID NO: 11), and Slit2 D1-E6 (SEQ ID NO: 12), as well as
derivative, analogs and homologs thereof capable of one or more of
the following: promoting endothelial barrier function in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibiting vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promoting the presence of VE-cadherin
at the surface of vascular endothelial cells; and promoting
expression of p120-catenin at the surface of vascular endothelial
cells.
[0067] In still other embodiments, where the active agent is
selected from a derivative, analog or homolog of one of the Slit2
polypeptides described herein, the active agent may be selected
from a derivative, analog or homolog of a naturally occurring
mammalian Slit2 polypeptide, or one of the Slit2 polypeptides
described by SEQ. ID. NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4 through
SEQ ID NO: 12, with such active agent exhibiting a polypeptide
sequence homology of at least about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or greater, to the relevant naturally occurring mammalian
Slit2 polypeptide, or to any one of SEQ. ID. NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 4 through SEQ ID NO: 12. In each such embodiment,
the derivative, analog or homolog is selected for its capacity for
one or more of the following: promoting endothelial barrier
function in the presence of one or more mediators of inflammation,
including one or more of an endotoxin (e.g., LPS), tumor necrosis
factor (e.g., TNF-.alpha.), and IL-1.beta.; inhibiting vascular
leak in the presence of one or more mediators of inflammation,
including one or more of an endotoxin (e.g., LPS), tumor necrosis
factor (e.g., TNF-.alpha.), and IL-1.beta.; promoting of the
presence of VE-cadherin at the surface of vascular endothelial
cells; and promoting expression of p120-catenin at the surface of
vascular endothelial cells
[0068] In another embodiment, the active agent is a derivative,
homolog, or analog of a naturally occurring mammalian Slit2
polypeptide, or one of the Slit2 polypeptides described by SEQ. ID.
NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4 through SEQ ID NO: 12, yet
exhibits less polypeptide sequence homology to the polypeptide from
which it is derived, such as, for example, a homology selected from
one of 80% or less, 70% or less, 60% or less, or 50% or less, while
retaining the capacity for one or more of the following: promoting
endothelial barrier function in the presence of one or more
mediators of inflammation, including one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.; inhibiting vascular leak in the presence of one or more
mediators of inflammation, including one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.; promoting the presence of VE-cadherin at the surface of
vascular endothelial cells; and promoting expression of
p120-catenin at the surface of vascular endothelial cells.
[0069] In one embodiment, an active agent as described herein is a
ligand of a Robo4 receptor. In such an embodiment, the ligand of
Robo4 can be any molecule that acts through Robo4 to promote
vascular barrier function. As used herein, the expression acts
through refers to a ligand that has an effect on endothelial cells
which requires the presence of the Robo4 receptor. In one
embodiment, a ligand effecting endothelial cells may act through
Robo4 by binding or associating with a Robo4 receptor in a manner
that results in Robo4 signaling. Without being bound by a
particular theory, it is presently believed that the Slit2
polypeptides described herein act through the Robo4 receptor.
Therefore, in specific embodiments where an active agent according
to the present description is a ligand of the Robo4 receptor, the
active agent may be selected from a Slit polypeptide described
herein. In another such embodiment, the ligand of Robo4 can be any
molecule that acts through Robo4 to promote the presence of
VE-cadherin at cell surface junctions. In specific embodiments, the
Slit ligand, or fragment or variant thereof, binds to or associates
with Robo4 in a manner that results in one or more of the
following: promotion of endothelial barrier function in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibition of vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promotion of the presence of
VE-cadherin at the surface of vascular endothelial cells; and
promotion of expression of p120-catenin at the surface of vascular
endothelial cells.
[0070] In yet another embodiment, an active agent as described
herein may be a ligand of Robo4, wherein the ligand acts through
Robo4 to promote cell surface expression of p120-catenin. In still
a further embodiment, an active agent as described herein includes
a ligand of a Robo4 receptor, wherein the ligand acts through Robo4
to initiate paxillin activation of GIT1. In another embodiment, an
active agent as described herein includes a ligand of a Robo4
receptor, wherein the ligand acts through Robo4 to activate GIT1
inhibition of ARF6. In a further embodiment, an active agent as
described herein includes a ligand of a Robo4 receptor, wherein the
ligand acts through Robo4 in a manner that results in one or more
of the following: promotion of endothelial barrier function in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibition of vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promotion of the presence of
VE-cadherin at the surface of vascular endothelial cells; and
promotion of expression of p120-catenin at the surface of vascular
endothelial cells. Where the active agent the present invention
includes a ligand of Robo4, in specific embodiments, the ligand can
be a molecule that binds the extracellular domain of Robo4 leading
to Robo4 signaling.
[0071] A polypeptide of a desired structure can be produced using
methods and materials well known in the art. For example, various
methods for isolating naturally occurring polypeptides or producing
recombinant polypeptides are well known. Moreover, various methods
are known for synthetically producing a polypeptide of desired
sequence. For example, peptides can be chemically synthesized using
currently available laboratory equipment using either Fmoc
(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)
chemistry. (Applied Biosystems, Inc., Foster City, Calif.). Slit
polypeptides described herein can be obtained through recombinant
and synthetic techniques well-known to those of skill in the art,
including those described herein and, for example, the methods
described in International Publication No. WO 2009/129408,
International Publication No. WO 2008/073441.
[0072] One skilled in the art can readily appreciate that a peptide
corresponding to a desired protein can be synthesized by standard
chemical reactions. For example, a peptide can be synthesized and
not cleaved from its synthesis resin whereas another peptide
fragment of a protein can be synthesized and subsequently cleaved
from the resin, thereby exposing a terminal group which is
functionally blocked on the other fragment. By peptide condensation
reactions, these two fragments can be covalently joined via a
peptide bond at their carboxyl and amino termini, respectively, to
form an antibody, or fragment thereof. (Grant G A (1992) Synthetic
Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky
M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-Verlag Inc., NY (which is herein incorporated by reference
at least for material related to peptide synthesis). Alternatively,
a desired protein or peptide can be synthesized in-vivo using
standard recombinant techniques. Where independent peptides that
are to be linked to form a desired protein are independently
produced in-vivo, once such independent peptides are produced and
isolated, they may be linked to form a desired protein or fragment
thereof via similar peptide condensation reactions.
[0073] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains. (Abrahmsen L et al., Biochemistry, 30:4151
(1991)). Alternatively, native chemical ligation of synthetic
peptides can be utilized to synthetically construct large peptides
or polypeptides from shorter peptide fragments. This method
consists of a two step chemical reaction. (Dawson et al. Synthesis
of Proteins by Native Chemical Ligation. Science, 266:776-779
(1994)). The first step is the chemoselective reaction of an
unprotected synthetic peptide-thioester with another unprotected
peptide segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site. (Baggiolini M et al.
(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
[0074] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
[0075] In another embodiment, an active agent as described herein
may be a small molecule active agent that inhibits the activity of
a cytohesin selected from the ARNO family of cytohesins. Small
molecule active agents selected from compounds that inhibit the
availability, activation or activity of an ARF-GEF, such as a
cytohesin, a cytohesin selected from the ARNO family of cytohesins,
or ARNO, in a manner that results in inhibition of one or more
ARFs, such as ARF6 and ARF1 are described in Jones et al.,
International Publication No. WO 2009/129408, and International
Publication No. WO 2008/073441. In the context of the compositions
and methods described herein, it has been determined that small
molecule active agents that inhibit the availability, activation or
activity of an ARF-GEF, such as a cytohesin, a cytohesin selected
from the ARNO family of cytohesins, or ARNO, in a manner that
results in inhibition of one or more ARFs, such as ARF6 and ARF1,
can inhibit pulmonary vascular permeability and/or inflammation and
the pulmonary fibrosis that can develop as a result.
[0076] In specific embodiments, a small molecule active agent as
described herein inhibits the activity of a cytohesin selected from
the ARNO family of cytohesins in a manner that results in one or
more of the following: inhibition of the activity or availability
of ARF6; inhibition of the activity or availability of ARF1;
promotion of vascular endothelial barrier function in the presence
of one or more mediators of inflammation, including one or more of
an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibition of vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promotion of the presence of
VE-cadherin at the surface of vascular endothelial cells; and
promotion of expression of p120-catenin at the surface of vascular
endothelial cells. In another embodiment, the small molecule active
agent inhibits the activity of ARNO in a manner that results in one
or more of the following: inhibition of ARF6; preservation of
vascular endothelial barrier function; promotion of endothelial
barrier function in the presence of one or more mediators of
inflammation, including one or more of an endotoxin (e.g., LPS),
tumor necrosis factor (e.g., TNF-.alpha.), and IL-1.beta.;
inhibition of vascular leak in the presence of one or more
mediators of inflammation, including one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.; promotion of the presence of VE-cadherin at the surface
of vascular endothelial cells; and promotion of expression of
p120-catenin at the surface of vascular endothelial cells. In yet
another embodiment, the small molecule active agent inhibits the
activity or availability of ARF6, resulting in one or more the
following: preservation of vascular endothelial barrier function;
promotion of endothelial barrier function in the presence of one or
more mediators of inflammation, including one or more of an
endotoxin (e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.),
and IL-1.beta.; inhibition of vascular leak in the presence of one
or more mediators of inflammation, including one or more of an
endotoxin (e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.),
and IL-1.beta.; promotion of the presence of VE-cadherin at the
surface of vascular endothelial cells; and promotion of expression
of p120-catenin at the surface of vascular endothelial cells.
[0077] In a specific embodiment, an active agent as described
herein may be SecinH3, the structure of which is provided in FIG.
9. SecinH3 is an inhibitor of cytohesins (see, for example, Hafner
et al, Inhibition of cytohesins by SecinH3 leads to hepatic insulin
resistance, Nature (2006), 444, 941-944, and International Patent
App. Publication No. WO 2006/053903, the contents of both of which
are incorporated herein by reference). It has been found that
Secin-H3 inhibits the effects of mediators of inflammation and
vascular permeability. Thus, in one embodiment, SecinH3 may be
selected as a small molecule active agent that inhibits the
activity of a cytohesin selected from the ARNO family of cytohesins
in a manner that results in inhibition of an ARF selected from ARF6
and ARF1, and provides one or more of the following: promotion of
endothelial barrier function in the presence of one or more
mediators of inflammation, including one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.; inhibition of vascular leak in the presence of one or
more mediators of inflammation, including one or more of an
endotoxin (e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.),
and IL-1.beta.; promotion of the presence of VE-cadherin at the
surface of vascular endothelial cells; and promotion of expression
of p120-catenin at the surface of vascular endothelial cells.
[0078] In another embodiment, a composition as described herein
includes one or more small molecule active agents selected from
compounds that inhibit the availability, activation or activity of
an ARF-GEF, such as a cytohesin, a cytohesin selected from the ARNO
family of cytohesins, or ARNO in a manner that results in one or
more of the following: inhibition of the activity or availability
of ARF6; inhibition of the activity or availability of ARF1;
preservation of vascular endothelial barrier function; promotion of
endothelial barrier function in the presence of one or more
mediators of inflammation, including one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.; inhibition of vascular leak in the presence of one or
more mediators of inflammation, including one or more of an
endotoxin (e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.),
and IL-1.beta.; promotion of the presence of VE-cadherin at the
surface of vascular endothelial cells; and promotion of expression
of p120-catenin at the surface of vascular endothelial cells.
[0079] Where an active agent according to the present description
includes a small molecule active agent, in specific embodiments,
the active agent may include one or more compounds having the
following chemical formula (Formula 1):
##STR00001##
[0080] wherein:
[0081] R.sup.1 and R.sup.3 are independently chosen from optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted cycloalkyl, or optionally substituted heterocycle;
[0082] R.sup.2 is chosen from hydrogen, lower alkoxy, lower alkyl,
halogen or hydroxy;
[0083] Z is chosen from O, S, NH, alkylene or a single bond; or
[0084] pharmaceutically acceptable salts, solvates or hydrates
thereof.
In one such embodiment, the one or more compounds are selected from
compounds as described by Formula 1, wherein R.sup.3 is substituted
with 1 to 5 substituents independently chosen from halogen, lower
alkyl, lower alkoxy, heteroatom lower alkyl, hydroxy, or methylene
dioxy, wherein two substituents together may form a fused
cycloalkyl or heterocyclic ring structure. In another such
embodiment, the one or more compounds are selected from compounds
as described by Formula 1, wherein R.sup.1 is chosen from
unsubstituted aryl or unsubstituted heteroaryl; R.sup.2 is chosen
from hydrogen, lower alkoxy, or lower alkyl; R.sup.3 is chosen from
aryl, optionally substituted with 1 to 5 substituents independently
chosen from halogen, lower alkyl, lower alkoxy, or methylene dioxy,
wherein two substituents together may form a fused cycloalkyl or
heterocyclic ring structure; and Z is chosen from O, S, or a single
bond.
[0085] In another embodiment, where an active agent according to
the present description includes a small molecule active agent, the
active agent may be selected from one or more compounds having the
following chemical formula (Formula 2):
##STR00002##
[0086] wherein:
[0087] R.sup.1 is chosen from optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted
cycloalkyl, or optionally substituted heterocycle;
[0088] R.sup.2 is chosen from hydrogen, lower alkoxy, lower alkyl,
halogen or hydroxy;
[0089] Z is chosen from O, S, NH, alkylene or a single bond;
[0090] X is independently chosen from halogen, lower alkyl, lower
alkoxy, heteroatom lower alkyl, hydroxy, or methylene dioxy,
wherein two substituents together may form a fused cycloalkyl or
heterocyclic ring structure;
[0091] m is 0 to 5; or
[0092] pharmaceutically acceptable salts, solvates or hydrates
thereof.
In one such embodiment, the one or more compounds are selected from
the following compounds:
##STR00003##
[0093] or pharmaceutically acceptable salts, solvates or hydrates
thereof.
[0094] As described in Jones et al., International Publication No.
WO 2009/129408, and International Publication No. WO 2008/073441,
it is believed that activation of Robo4 results in interaction
between Robo4 with the adaptor protein, paxillin, and its
paralogues, which recruits ARF-GAPs, such as GIT1, leading to local
inactivation of ARF6. It is further described in Jones et al.,
International Publication No. WO 2009/129408, and International
Publication No. WO 2008/073441, modulation of AFR-GAPs and ARF-GEFs
can be accomplished without Robo4 signaling. Without being bound by
a particular theory, it is presently believed that the small
molecule active agents described herein may function, at least in
part, by achieving the benefits of Robo4 signaling, such as those
described herein and in Jones et al., International Publication No.
WO 2009/129408, and International Publication No. WO 2008/073441,
without requiring the use of a Robo4 ligand or direct activation of
Robo4.
[0095] Compositions including an active agent as described herein
are also provided. Such compositions may include one or more active
agents as described herein. In one embodiment, a composition is
prepared as a pharmaceutical formulation. For example, in addition
to one or more active agent as described herein, a pharmaceutical
formulation may include a pharmaceutically acceptable carrier
and/or one or more pharmaceutically acceptable excipients to
provide a formulation that is suitable for therapeutic
administration. As used herein, pharmaceutically acceptable refers
to a material that is not biologically or otherwise undesirable,
e.g., the material is suitable for administration to a subject
together with the desired active agent (e.g., a desired active
agent as described herein) and is compatible with other components
of the pharmaceutical formulation in which it is contained. The
carrier and any excipient(s) would naturally be selected to
minimize any degradation of the active agent or adverse side
effects in the subject.
[0096] A pharmaceutical formulation according to the present
description may be prepared in any form suitable for
administration, such as, by way of example, a tableted composition,
a powder composition for encapsulation, a solution composition for
direct ingestion, encapsulation or parenteral delivery, an
emulsion, a gel, a cream, suppository, or a suspension, such as a
formulation that incorporates or is incorporated into, for example,
microparticles, a matrix material, or liposomes. A pharmaceutical
formulation as described herein may include components targeted to
a particular cell type via antibodies, receptors, or receptor
ligands. Pharmaceutical carriers and excipients and their
formulations are well described in the literature, including, for
example, in Remington: The Science and Practice of Pharmacy (19th
ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa.
1995.
[0097] Where appropriate, a pharmaceutically-acceptable salt or
other tonicity modifying agent may be used in the pharmaceutical
formulation to render the formulation isotonic. Examples of liquid
pharmaceutically-acceptable carriers include, but are not limited
to, saline, Ringer's solution, and dextrose solution. Where the
pharmaceutical formulation is provided as a solution or suspension,
particularly for parenteral delivery, the pH of the formulation can
be adjusted as desired to facilitate delivery to a subject and/or
preservation of the active agent or other formulation components.
Carriers and excipients suitable for preparing pharmaceutical
formulations include, for example, a well-known variety of
pharmaceutically acceptable polymers, saccharides, salts, lipids,
phospholipids, surfactants, gels, polypeptides, and amino acids.
The pharmaceutical formulation according to the present description
may include sustained release preparations. It will be apparent to
those persons skilled in the art that certain carriers and/or
excipients may be more preferable depending upon, for instance, the
route of administration and concentration of composition being
administered. A pharmaceutical formulation as described herein may
include one or more thickener, flavoring, diluent, buffer,
preservative, antimicrobial agents, antiinflammatory agents,
anesthetics, surface active agent, and the like.
[0098] The active agents and compositions may be administered as a
pharmaceutically acceptable acid- or base-addition salt. Where that
is the case, the desired salt may be formed by reaction with an
inorganic acid, such as hydrochloric acid, hydrobromic acid,
perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and
phosphoric acid, and organic acids, such as formic acid, acetic
acid, propionic acid, glycolic acid, lactic acid, pyruvic acid,
oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric
acid, or by reaction with an inorganic base, such as sodium
hydroxyide, ammonium hydroxide, potassium hydroxide, and organic
bases such as mono-, di-, trialkyl and aryl amines and substituted
ethanolamines.
[0099] The compositions disclosed herein, including pharmaceutical
formulations, may be administered in a number of ways depending on
whether local or systemic treatment is desired, and on the area to
be treated. Parenteral administration of the composition, if used,
is generally characterized by injection. Injectables can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for dissolution or suspension in
liquid prior to injection, or as emulsions. A revised approach for
parenteral administration involves use of a slow release or
sustained release system such that a constant dosage is maintained.
(See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by
reference herein.)
[0100] The exact amount of a given composition required to achieve
a therapeutic affect will vary from subject to subject, depending
on the species, age, weight and general condition of the subject,
the severity of the pathologic condition being treated, the
particular active agent used, its mode of administration, and the
like. The dosage ranges for the administration of the compositions
are those large enough to produce a therapeutic effect. The dosage
can be adjusted to avoid or reduce the occurrence of adverse side
effects, such as unwanted cross-reactions, anaphylactic reactions,
and the like. The dosage may vary with the age, condition, sex and
extent of the disease in the patient, route of administration, or
whether other drugs are included in the regimen. The dosage can be
adjusted by the individual physician in the event of any counter
indications. Dosage can vary, and can be administered in one or
more dose administrations daily, for one or several days. Guidance
can be found in the literature for appropriate dosages for given
classes of pharmaceutical products.
III. Methods
[0101] Methods for promoting vascular endothelial barrier function
are provided herein. In one embodiment, a method for promoting
vascular endothelial barrier function includes treating one or more
vascular endothelial cells with an active agent as described
herein. In one such embodiment, the step of treating one or more
vascular endothelial cells may be carried out by administering to a
patient in need thereof a therapeutically effective amount of an
active agent as described herein. Where desired, the active agent
may be administered using a composition as described herein. In
particular embodiments, treatment of the one or more vascular
endothelial cells with the active agent results in one or more of
the following: preservation of vascular endothelial barrier
function; promotion of endothelial barrier function in the presence
of one or more mediators of inflammation, including one or more of
an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; inhibition of vascular leak in the
presence of one or more mediators of inflammation, including one or
more of an endotoxin (e.g., LPS), tumor necrosis factor (e.g.,
TNF-.alpha.), and IL-1.beta.; promotion of the presence of
VE-cadherin at the surface of vascular endothelial cells; and
promotion of expression of p120-catenin at the surface of vascular
endothelial cell. In one such embodiment, treatment of the one or
more vascular endothelial cells with the active agent enhances the
presence of VE-cadherin at the surface of vascular endothelial
cells and promotes expression of p120-catenin at the surface of
vascular endothelial cells. In another such embodiment, treatment
of the one or more vascular endothelial cells with the active agent
restores, at least in part, vascular barrier function after
exposure of the vascular endothelial cells to one or more mediators
of inflammation, wherein the one or more mediators of inflammation
are selected from including one or more of an endotoxin (e.g.,
LPS), tumor necrosis factor (e.g., TNF-.alpha.), and IL-1.beta..
The active agent may be selected from the active agents described
herein and administration of the active agent may be accomplished
by administration of such active agent using a composition as
described herein.
[0102] The methods for promoting vascular endothelial barrier
function described herein may be utilized for promoting barrier
function in various different endothelial tissues, including
endothelial tissues selected from one of vascular endothelium of
the lung, vascular endothelium of the kidney and vascular
endothelium of the spleen. Therefore, in specific embodiments,
treating one or more endothelial cells with an active agent as
described herein may include treating vascular endothelial cells
selected from vascular endothelial cells of the lung, vascular
endothelial cells of the kidney, and vascular endothelial cells of
the spleen.
[0103] In another embodiment, the methods of the present invention
include treating a patient at risk for or suffering from acute
pulmonary vascular edema, chronic pulmonary vascular edema, acute
pulmonary vascular inflammation, chronic pulmonary vascular
inflammation, pulmonary fibrosis, including idiopathic pulmonary
fibrosis, bacterial sepsis, or influenza infection, such as an
avian flu infection. Therefore, in particular embodiments, the
methods of the present invention include identifying a patient at
risk of or suffering from one or more of acute pulmonary vascular
edema, chronic pulmonary vascular edema, acute pulmonary vascular
inflammation, chronic pulmonary vascular inflammation, pulmonary
fibrosis, including idiopathic pulmonary fibrosis, bacterial
sepsis, or influenza infection, such as an avian flu infection, and
administering to the patient a therapeutically effective amount of
an active agent as described herein. Where desired, the active
agent may be administered using a composition as described herein.
In specific embodiments, administration of the active agent results
in one or more of the following: promotion of endothelial barrier
function in the presence of one or more mediators of inflammation,
including one or more of an endotoxin (e.g., LPS), tumor necrosis
factor (e.g., TNF-.alpha.), and IL-1.beta.; inhibition of vascular
leak in the presence of one or more mediators of inflammation,
including one or more of an endotoxin (e.g., LPS), tumor necrosis
factor (e.g., TNF-.alpha.), and IL-1.beta.; promotion of the
presence of VE-cadherin at the surface of vascular endothelial
cells; and promotion of expression of p120-catenin at the surface
of vascular endothelial cells.
[0104] In another embodiment, the methods of the present invention
include restoring vascular endothelial barrier function in a
patient, wherein the patient is suffering from a pathologic
condition selected from acute pulmonary vascular edema, chronic
pulmonary vascular edema, acute pulmonary vascular inflammation,
chronic pulmonary vascular inflammation, pulmonary fibrosis,
including idiopathic pulmonary fibrosis, bacterial sepsis, or
influenza infection, such as an avian flu infection. Moreover, the
pathologic condition or environmental condition may be further
associated with the presence or expression of one or more mediators
of inflammation, such as for example, one or more of an endotoxin
(e.g., LPS), tumor necrosis factor (e.g., TNF-.alpha.), and
IL-1.beta.. In a method for restoring vascular barrier function as
described herein, a therapeutically effective amount of an active
agent as described herein is administered to the patient. Where
desired, the active agent may be administered using a composition
as described herein. In specific embodiments, administration of the
active agent results in one or more of the following: restoration
of vascular barrier function; inhibition of vascular leak; enhanced
presence of VE-cadherin at the surface of vascular endothelial
cells; and enhanced expression of p120-catenin at the surface of
vascular endothelial cells.
[0105] In yet further embodiments, the methods of the present
invention include methods for promoting the presence of VE-cadherin
at the surface of vascular endothelial cells. In particular
embodiments, a method for promoting the presence of VE-cadherin at
the surface of vascular endothelial cells includes treating one or
more vascular endothelial cells with an active agent as described
herein. In one such embodiment, the step of treating one or more
vascular endothelial cells may be carried out by administering to a
patient in need thereof a therapeutically effective amount of an
active agent as described herein. Where desired, the active agent
may be administered using a composition as described herein. In
particular embodiments, treatment of the one or more vascular
endothelial cells with the active agent results in one or both of
promoting the presence of VE-cadherin at the surface of vascular
endothelial cells and promoting expression of p120-catenin at the
surface of vascular endothelial cells. In one such embodiment,
treatment of the one or more vascular endothelial cells with the
active agent enhances the presence of VE-cadherin at the surface of
vascular endothelial cells and promotes expression of p120-catenin
at the surface of vascular endothelial cells.
[0106] Methods of screening for or evaluating an agent that
promotes vascular endothelial barrier function are also provided
herein. For example, in particular embodiments, methods of
screening for active agents according to the present description
can be carried out using the in-vitro experiments and in-vivo
models described herein. In a specific embodiment of a method of
screening active agents as described herein, the method may include
evaluating the ability of an active agent to promote the presence
of VE-cadherin at the surface of vascular endothelial cells
utilizing the experimental protocols provided herein. In another
embodiment of a method of screening active agents as described
herein, the method may include evaluating the ability of an active
agent to promote expression of p120-catenin at the surface of
vascular endothelial cells utilizing the experimental protocols
provided herein. In yet a further embodiment, a method of screening
active agents as described herein may include evaluating the
ability of an active agent to preserve endothelial barrier function
utilizing the experimental protocols provided herein. In still a
further embodiment, a method of screening active agents may include
evaluating the ability of an active agent to inhibit formation of
pulmonary fibrosis in an animal model of Bleomycin-induced
fibrosis, as described herein.
[0107] In a specific embodiment, a method for identifying an agent
inhibits the activity or availability of a targeted ARF-GEF, such
as a cytohesin, a cytohesin selected from the ARNO family of
cytohesins, or ARNO, in a manner that results in inhibition the
activity or availability of one or more ARFs, such as ARF6 and
ARF1, involves an aptamer-displacement screen assay as described,
for example, by Hafner et al. (Displacement of protein-bound
aptamers with small molecules screened by fluorescence
polarization, Nat Protoc (2008), 3, 579-587). In particular, such a
method can be used to identify and confirm the activity of small
molecules, such as those described herein. The association of the
aptamer with its target is detected by fluorescence polarization.
The fluorescence-labeled aptamer exhibits low polarization in the
non-bound state. When bound to the target protein, the fluorescence
polarization of the fluorescence-labeled aptamer is increased. If a
small molecule displaces the aptamer from the protein, the
fluorescence polarization of the fluorescence-labeled aptamer
decreases, thereby allowing identification of small molecule
candidates exhibiting activities analogous to the fluorescence
labeled aptamer.
IV. Examples
[0108] The Examples that follow are offered for illustrative
purposes only and are not intended to limit the scope of the
compositions and methods described herein in any way. It is to be
understood that the disclosed compositions and methods are not
limited to the particular methodologies, protocols, and reagents
described herein. In each instance, unless otherwise specified,
standard materials and methods were used in carrying out the work
described in the Examples provided. All patent and literature
references cited in the present specification are hereby
incorporated by reference in their entirety.
[0109] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA, genetics,
immunology, cell biology, cell culture and transgenic biology,
which are within the skill of the art. (See, e.g., Maniatis, T., et
al. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.); Sambrook, J., et al.
(1989) Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed. (Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); Ausubel, F.
M., et al. (1992) Current Protocols in Molecular Biology, (J. Wiley
and Sons, NY); Glover, D. (1985) DNA Cloning, I and II (Oxford
Press); Anand, R. (1992) Techniques for the Analysis of Complex
Genomes, (Academic Press); Guthrie, G. and Fink, G. R. (1991) Guide
to Yeast Genetics and Molecular Biology (Academic Press); Harlow
and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.); Jakoby, W. B. and Pastan, I.
H. (eds.) (1979) Cell Culture. Methods in Enzymology, Vol. 58
(Academic Press, Inc., Harcourt Brace Jovanovich (NY); Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Hogan et al. (eds) (1994)
Manipulating the Mouse Embryo; A Laboratory Manual, 2.sup.nd
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.). A general discussion of techniques and materials for human
gene mapping, including mapping of human chromosome 1, is provided,
e.g., in White and Lalouel (1988) Ann. Rev. Genet. 22:259 279. The
practice of the present invention employs, unless otherwise
indicated, conventional techniques of chemistry, molecular biology,
microbiology, recombinant DNA, genetics, and immunology. (See,
e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al.,
1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991).
Nothing herein is to be construed as an admission that the subject
matter taught herein is not entitled to antedate such disclosure by
virtue of prior invention. No admission is made that any reference
constitutes prior art.
Example 1
Slit-Robo4 Signaling Reduces Endothelial Hyperpermeability Induced
by Multiple Mediators of Inflammation
[0110] Slit-Robo4 signaling reduces endothelial hyperpermeability
induced by endotoxin (lipopolysaccharide, LPS), tumor necrosis
factor-.alpha. (TNF-.alpha.), and interleukin-1.beta. (IL-1.beta.),
all important mediators of inflammation (Dinarello, C. A. 1997.
Proinflammatory and anti-inflammatory cytokines as mediators in the
pathogenesis of septic shock. Chest 112:321 S-329S). To study
barrier function in vitro, we assessed the ability of a human
endothelial cell monolayer to act as a barrier to diffusion of a
horseradish peroxidase (HRP) reporter. We utilized the N-terminal
fragment (Slit2N), which is the active fragment of Slit that is
released by proteolytic cleavage (Chedotal, A. 2007. Slits and
their receptors. Adv Exp Med Biol 621:65-80). As shown in FIG. 1a,
Slit2N significantly reduced LPS, TNF-.alpha., and IL-1.beta.
induced permeability. Furthermore, the inhibitory effect of Slit2N
was lost in cells exposed to siRNA directed against Robo4 (FIG. 1B;
FIG. 14A).
Example 2
Slit2-Robo4 Promotes Vascular Stability by Directly Enhancing the
Machinery Responsible for Cell-Cell Interactions
[0111] The Slit2-Robo4 pathway promotes vascular stability by
directly enhancing the machinery responsible for cell-cell
interactions. In the endothelium, critical stabilizing interactions
are mediated by the adherens junction protein, vascular endothelial
cadherin (VE-cadherin) (Dejana, E., F. Orsenigo, and M. G.
Lampugnani. 2008. The role of adherens junctions and VE-cadherin in
the control of vascular permeability. J Cell Sci 121:2115-2122; and
Vestweber, D. 2008. VE-cadherin: the major endothelial adhesion
molecule controlling cellular junctions and blood vessel formation.
Arterioscler Thromb Vasc Biol 28:223-232). We found that treating
human microvascular lung endothelial cells (HMVEC-lung) with Slit2N
significantly increased VE-cadherin levels at the cell surface
junctions (FIG. 1C, F; FIG. 14B). VE-cadherin surface expression is
regulated by the association of p120-catenin with VE-cadherin, an
association known to inhibit VE-cadherin internalization from the
cell surface and promote vascular stability (Potter, M. D., S.
Barbero, and D. A. Cheresh. 2005. Tyrosine phosphorylation of
VE-cadherin prevents binding of p120- and beta-catenin and
maintains the cellular mesenchymal state. J Biol Chem
280:31906-31912; and Xiao, K., J. Garner, K. M. Buckley, P. A.
Vincent, C. M. Chiasson, E. Dejana, V. Faundez, and A. P.
Kowalczyk. 2005. p120-Catenin regulates clathrin-dependent
endocytosis of VE-cadherin. Mol Biol Cell 16:5141-5151). Slit2N
also increased cell surface p120-catenin expression (FIG. 1D), but
had no observed effect on other junction or catenin family members
(FIG. 1E, data not shown).
Example 3
Slit2 Enhances VE-Cadherin at the Cell Surface Following Exposure
to IL-1.beta.
[0112] IL-1.beta. reduces VE-cadherin levels at the cell surface
and Slit2N negated this effect (FIG. 2A). IL-1.beta. stimulation
decreased p120-catenin at the cell surface and Slit2N reversed this
effect (FIG. 2A). IL-1.beta.-induced dissociation of VE-cadherin
from p120-catenin and internalization of VE-cadherin (FIG. 2B, C).
Slit2N restores association of VE-cadherin and p120-catenin, and
blocks internalization of VE-cadherin (FIG. 2B, C). To investigate
whether the effect of Slit2N on VE-cadherin localization is
necessary for its ability to enhance vascular stability, we
examined if an anti-VE-cadherin antibody could block the effect of
Slit2N on permeability in vitro. Slit2N inhibited
IL-1.beta.-induced permeability in vitro in the presence of a
non-specific IgG; however, the effect of Slit2N was lost in the
presence of an anti-VE-cadherin antibody (FIG. 2D). Together, these
data demonstrate that Slit preserves the association of
p120-catenin with VE-cadherin in the face of IL-1.beta.
stimulation, thereby promoting vascular integrity by reducing
cytokine induced VE-cadherin endocytosis.
Example 4
Slit2 Reduces Vascular Permeability In-Vivo Under Conditions of
Cytokine Storm
[0113] To illustrate that Slit reduces vascular permeability
in-vivo under conditions that result in cytokine storm, we utilized
a bacterial endotoxin model of pulmonary inflammation. In this
model, lipopolysaccharide (LPS) is administered to the lungs of
mice through intratracheal instillation, simulating a gram-negative
infection (Matute-Bello, G., C. W. Frevert, and T. R. Martin. 2008.
Animal models of acute lung injury. Am J Physiol Lung Cell Mol
Physiol 295:L379-399). LPS instillation in the lung is a model of
acute inflammation. The administration of LPS triggers a massive
inflammatory reaction and release of cytokines, resulting in a
significant increase in alveolar capillary permeability.
[0114] Using Evans Blue Albumin (EBA) as a tracer, we found that
Slit2N significantly reduces vascular leak in the lungs of LPS
treated Robo4.sup.+/+ mice (FIG. 3A). The effect of Slit2N was lost
in Robo4-null (Robo4.sup.AP/AP) mice, demonstrating that Robo4 is
necessary for the effect of Slit2N in vivo (FIG. 3A). This result
also indicates that this activity is endothelial-specific, as Robo4
is only detected in the endothelium (Huminiecki, L., M. Gorn, S.
Suchting, R. Poulsom, and R. Bicknell. 2002. Magic roundabout is a
new member of the roundabout receptor family that is endothelial
specific and expressed at sites of active angiogenesis. Genomics
79:547-552; and Park, K. W., C. M. Morrison, L. K. Sorensen, C. A.
Jones, Y. Rao, C. B. Chien, J. Y. Wu, L. D. Urness, and D. Y. Li.
2003. Robo4 is a vascular-specific receptor that inhibits
endothelial migration. Dev Biol 261:251-267). LPS instillation in
the lung also induces accumulation of protein exudates and
leukocytes in the alveolar space, inflammatory responses that can
be quantified by bronchoalveolar lavage (BAL) (Matute-Bello et al.,
2008). Slit2N reduced protein exudates, a key marker of acute lung
injury and indicator of vascular barrier disruption (Ware, L. B.,
and M. A. Matthay. 2000. The acute respiratory distress syndrome. N
Engl J Med 342:1334-1349), and inflammatory cell accumulation in
the bronchoalveolar lavage fluid (BALF) of Robo4.sup.+/+ mice in a
dose-dependent manner (FIG. 3B-D; FIG. 14C, D). Inhibition of
protein and leukocyte accumulation in BALF was lost in
Robo4.sup.AP/AP mice, indicating again that Slit2N acts directly
upon the vasculature to decrease protein exudates and inflammatory
cell accumulation in the alveoli (FIG. 3B-D). Finally, histological
examination of the lung confirmed that Slit2N acts in a
Robo4-dependent manner by reducing LPS-induced lung inflammation in
Robo4.sup.+/+ but not Robo4.sup.AP/AP mice (FIG. 3E; FIG. 15). We
confirmed that the pulmonary vasculature of Robo4.sup.AP/AP mice
developed normally (FIGS. 19 and 20), indicating that the loss of
the effect of Slit2 in Robo4.sup.AP/AP was not due to structural
differences in the vasculature.
[0115] As neutrophils are a predominant cell type in bacterial
pneumonia and LPS challenge models (Matute-Bello et al., 2008), we
asked if Slit2N had a direct effect on neutrophil migration. It has
previously been reported that Slit2 has a profound impact on
neutrophil migration (Wu, J., et al. The neuronal repellent Slit
inhibits leukocyte chemotaxis induced by chemotactic factors.
Nature 410, 948-952 (2001). However, these experiments were done
using DMSO-treated HL-60 neutrophil-like cells. We observed the
same effect of Slit2 on DMSO-treated HL-60 neutrophil-like cells,
but found primary human PMNs did not respond to Slit2N, which is
consistent with the fact that they do not express Robo receptors
(FIG. 16A, B).
[0116] In initial studies, we did not detect enhanced sensitivity
in the lungs of Robo4.sup.AP/AP mice compared to Robo4.sup.+/+ mice
in the LPS-model of acute inflammation (FIG. 3B-D). However, using
quantitative PCR, we found that Robo4 expression was significantly
increased in the lung 6 hours after LPS instillation (FIG. 11).
This result suggested that Robo4 may be important for inhibiting or
resolving LPS-induced inflammation in the lung. We reasoned that if
the level of LPS administered was too high, it would cause such
severe damage that any difference between the two genotypes would
be masked. Thus, we lowered the dose of LPS used to challenge the
mice, and found that Robo4.sup.AP/AP mice had significantly higher
protein levels in BALF exudates compared to littermate
Robo4.sup.AP/AP mice (FIG. 3F). This increased sensitivity
indicates a role for endogenous Slit-Robo4 signaling in dampening
the vascular effects of cytokines Consistent with this model, Slit2
protein is expressed throughout the lung and in close proximity to
the endothelium (FIG. 17).
Example 5
Slit Signals Via a VE-Cadherin Dependent Mechanism In-Vivo
[0117] To confirm that Slit signals via a VE-cadherin dependent
mechanism in-vivo, we blocked VE-cadherin with a specific antibody
that prevents homophilic interactions between VE-cadherin expressed
on adjacent endothelial cells. Slit2N reduced protein exudates and
inflammatory cell infiltration in the presence of a control IgG
antibody, but not in the presence of a VE-cadherin blocking
antibody (FIG. 3G-I). Thus, similar to the results of cell culture
experiments, the in-vivo data support a model of Slit-Robo4
promoting VE-cadherin expression at the cell surface and blunting
of cytokine mediated endothelial hyperpermeability.
Example 6
Various Slit Proteins Work to Activate Robo4
[0118] FIG. 7 illustrates various constructs of the Slit2 protein.
As has already been described herein, the 150 kD protein Slit2N
(SEQ ID NO: 4), has been found to be effective in in vitro and in
vivo models, including Miles assays, assays for retinal
permeability, tube formation and endothelial cell migration and in
OIR and CNV models of ocular disease. In FIG. 7A, different
constructs for the Slit protein are depicted. The four leucine rich
domains (LRR), the epidermal growth factor homology region (EGF)
and the c-terminal tags (MYC/HIS) are indicated. Inhibition of VEGF
mediated endothelial cell migration by the different Slit
constructs (2 nM) is shown in FIG. 7B.
Example 7
Robo4 Knockout Mouse
[0119] The Robo4 knockout mice utilized in the experimental
examples detailed herein were produced using standard techniques.
To produce the knockout mice, exons one through five of the gene
expressing Robo4 were replaced with an alkaline phosphatase (AP)
reporter gene using homologous recombination. This allele,
Robo4.sup.AP, lacked the exons encoding the immunoglobulin (IgG)
repeats of the Robo4 ectodomain, which are predicted to be required
for interaction with Slit proteins. The Robo4.sup.+/AP animals were
intercrossed to generate mice that were homozygous for the targeted
allele. An illustration of the genomic structure of the mice is
provided in FIG. 12. Robo4.sup.AP/AP animals were viable and
fertile, and exhibited normal patterning of the vascular system.
These data indicate that Robo4 is not required for sprouting
angiogenesis in the developing mouse, and point to an alternate
function for Robo4 signaling in the mammalian endothelium. Alkaline
phosphatase activity was detected in these animals throughout the
endothelium of all vascular beds in the developing embryos and in
the adult mice, which confirmed that the Robo4.sup.AP allele is a
valid marker of Robo4 expression.
Example 8
Slit2 Reduces Vascular Permeability and Development of Pulmonary
Fibrosis In-Vivo
[0120] Slit2N reduces vascular permeability in-vivo in the setting
of chronic inflammation (Matute-Bello, G., Frevert, C., &
Martin, T. Animal models of acute lung injury. Am J Physiol Lung
Cell Mol Physiol 295, 379-399 (2008); Gasse, P., et al.
IL-1R1/MyD88 signaling and the inflammasome are essential in
pulmonary inflammation and fibrosis in mice. J Clin Invest 117,
3786-3799 (2007); Russo, R., et al. Role of the chemokine receptor
CXCR2 in bleomycin-induced pulmonary inflammation and fibrosis. Am
J Respir Cell Mol Biol (2008)). Bleomycin is a chemical that causes
prolonged and chronic permeability in the lung (Tager, A., et al.
The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to
lung injury by mediating fibroblast recruitment and vascular leak.
Nat Med 14, 45-54 (2008)). In addition to chronic permeability and
inflammation, Bleomycin causes pulmonary fibrosis. In Robo4.sup.+/+
mice, Slit2N significantly reduced Bleomycin-induced EBA
accumulation in the lung eleven days after Bleomycin administration
(FIG. 13a). The effect of Slit2N was lost in Robo4.sup.AP/AP mice
(FIG. 13a). Slit2N also significantly reduced Bleomycin-induced
pulmonary fibrosis (FIG. 13b). This effect was lost in
Robo4.sup.AP/AP mice, indicating that Slit2N acted directly upon
the endothelium to reduce pulmonary fibrosis (FIG. 13b). Histologic
examination of the lung using a trichrome stain to enhance the
visualization of collagen deposition confirmed the effect of Slit2N
in a Robo4-dependent manner (FIG. 13c). These results highlight the
value in of vascular stabilization in reducing pulmonary fibrosis
and underscore the role the endothelium plays in the pathogenesis
of fibrosis.
Example 9
SecinH3 Inhibits Bleomycin-Induced Fibrosis In-Vivo
[0121] The ability of SecinH3 to inhibit pulmonary fibrosis was
evaluated in an animal model of Bleomycin-induced fibrosis, as
described in Example 8. In brief, 6-8 week old mice were
anesthetized and given Bleomycin (0.05 U in 40 .mu.L saline) by
intranasal instillation. Control mice received an intranasal
instillation of 40 .mu.L saline. Mice were given an intraperitoneal
injection twice daily of 30 uM SecinH3 or vehicle. On day 11, mice
were sacrificed by CO.sub.2 asphyxiation, lungs removed, and
homogenized in 0.5M acetic acid with protease inhibitors (Roche).
Pulmonary collagen content was assessed using the Sircol collagen
assay. Homogenates were incubated overnight at 4.degree. C. with
stirring. Samples were then centrifuged and 1 mL Sircol dye reagent
was added to 100 .mu.L of supernatant for 30 minutes. Samples were
again centrifuged, the pellet re-suspended with 1 mL alkali
reagent, and analyzed by spectrophotometry (Biocolor). These
samples were compared against a collagen standard curve provided by
the manufacturer. Data are presented as s.e.m. of at least seven
mice per condition.
Example 10
Slit Enhances Vascular Stability During Polymicrobial Sepsis
[0122] Slit2N reduces mortality in the setting of systemic vascular
instability, and the effects of Slit2N are not limited to the lung.
In a model of polymicrobial sepsis known as cecal ligation and
puncture (CLP) (Hubbard, W. J., M. Choudhry, M. G. Schwacha, J. D.
Kerby, L. W. Rue, 3rd, K. I. Bland, and I. H. Chaudry. 2005. Cecal
ligation and puncture. Shock 24 Suppl 1:52-57). Slit2N
significantly reduced vascular permeability in the kidney and
spleen (FIG. 4A, B) and improved the survival of mice exposed to
CLP-induced sepsis from 33% to nearly 80% (FIG. 4C). Under the
conditions of these experiments, CLP did not cause significant
damage to the lung, providing further support that the effect of
Slit2N was not limited to the lung (FIG. 18A-C). Because a
hyper-inflammatory response contributes to the pathogenesis of
sepsis, we tested whether Slit2N affects cytokine and chemokine
levels in the plasma of septic mice (Dinarello, C. A. 1997.
Proinflammatory and anti-inflammatory cytokines as mediators in the
pathogenesis of septic shock. Chest 112:321 S-329S). Slit2N did not
alter the expression of a panel of cytokines and chemokines,
demonstrating that the therapeutic effect of Slit2N is not
secondary to a reduction in inflammatory cytokine and chemokine
levels (FIG. 4D, E). Finally, the effect of Slit2N was lost in
Robo4.sup.AP/AP mice (FIG. 4F), demonstrating that Robo4 is
necessary for the activity of Slit2N. Taken together, these data
demonstrate that Slit enhances survival during the systemic
inflammatory response triggered by sepsis by specifically enhancing
vascular stability.
Example 11
Slit Enhances Vascular Stability Resulting from Viral Infection
[0123] The effects of Slit were examined in a model of H5N1
influenza. Pandemic influenzas such as avian flu (H5N1) are
examples of lung injury caused by direct infection that are often
characterized by a massive increase in cytokine levels and
excessive inflammation (de Jong, M. D., C. P. Simmons, T. T. Thanh,
V. M. Hien, G. J. Smith, T. N. Chau, D. M. Hoang, N. V. Chau, T. H.
Khanh, V. C. Dong, P. T. Qui, B. V. Cam, Q. Ha do, Y. Guan, J. S.
Peiris, N. T. Chinh, T. T. Hien, and J. Farrar. 2006. Fatal outcome
of human influenza A (H5N1) is associated with high viral load and
hypercytokinemia. Nat Med 12:1203-1207; Kobasa, D., S. M. Jones, K.
Shinya, J. C. Kash, J. Copps, H. Ebihara, Y. Hatta, J. H. Kim, P.
Halfmann, M. Hatta, F. Feldmann, J. B. Alimonti, L. Fernando, Y.
Li, M. G. Katze, H. Feldmann, and Y. Kawaoka. 2007; Aberrant innate
immune response in lethal infection of macaques with the 1918
influenza virus. Nature 445:319-323; and Abdel-Ghafar, A. N., T.
Chotpitayasunondh, Z. Gao, F. G. Hayden, D. H. Nguyen, M. D. de
Jong, A. Naghdaliyev, J. S. Peiris, N. Shindo, S. Soeroso, and T.
M. Uyeki. 2008. Update on avian influenza A (H5N1) virus infection
in humans. N Engl J Med 358:261-273). Slit2N significantly
inhibited endothelial hyperpermeability in the lung three days
post-H5N1 infection (FIG. 5A) and reduced mortality (FIG. 5B). The
lung pathology in Slit2N treated mice was decreased in severity
compared to mock treated mice (FIG. 5C). To exclude the possibility
that Slit2N has a direct anti-viral activity, we measured lung
viral titers and found that Slit2N did not alter viral load (FIG.
5D); further, Slit2N does not significantly reduce the level of
inflammatory cytokine release following H5N1 infection (FIG. 5E,
F). Thus, the H5N1 results are consistent with our LPS and CLP
studies, and indicate that specifically limiting the vascular
response to hypercytokinemia is sufficient to reduce mortality and
morbidity in animal models of serious infections.
V. Materials & Methods
[0124] Preparation of recombinant Slit2N. 293T cells plated onto
Poly-L-lysine (Sigma) coated dishes were transiently transfected
with empty vector pSecTagB or pSecTagB::hSlit2N. For each 15 cm
dish of cells, 60 .mu.g DNA and 100 .mu.L Lipofectamine
(Invitrogen) in serum-free Opti-MEM was used. Slit2N protein was
salt extracted as previously described in Jones, C. A., N. R.
London, H. Chen, K. W. Park, D. Sauvaget, R. A. Stockton, J. D.
Wythe, W. Suh, F. Larrieu-Lahargue, Y. S. Mukouyama, P. Lindblom,
P. Seth, A. Frias, N. Nishiya, M. H. Ginsberg, H. Gerhardt, K.
Zhang, and D. Y. Li. 2008. Robo4 stabilizes the vascular network by
inhibiting pathologic angiogenesis and endothelial
hyperpermeability. Nat Med 14:448-453. Using this protocol, Slit2N
concentrations of 0.5-1.5 mg/mL were routinely obtained. We
performed the same salt extraction procedure on cells transfected
with empty vector pSecTagB. This preparation is referred to as Mock
and was used as a control for Slit2N in all experiments. In vitro
studies were conducted using 10 nM Slit2N.
[0125] LPS-induced acute lung injury. Eight to twelve week old
C57BL/6 mice were injected intravenously (IV) with saline alone or
3.5 .mu.g Slit2N or Mock in saline. Alternatively, the intravenous
injection also contained 20 .mu.g control IgG or 20 .mu.g
VE-cadherin blocking antibody (clone BV13, eBiosciences). Animals
were anesthetized with Avertin before surgical exposure of the
trachea. 10 .mu.g Lipopolysaccharide (serotype 0111:B4, Sigma) in
100 .mu.L saline or saline alone was administered intratracheally
(IT). Twenty four hours later, the trachea was re-exposed and
catheterized. Bronchoalveolar lavage fluid (BALF) was obtained by
injection of 1 mL saline followed by aspiration repeated three
times. BALF was centrifuged at 300 g for 5 minutes to recover
inflammatory cells. The pellet was treated with ACK buffer for 3
minutes to remove red blood cells. Cells were centrifuged at 300 g
for 5 minutes, and resuspended in 1 mL PBS containing 1% FBS. Cell
counts were then determined by hemocytometer. Neutrophil counts
were determined by cell differential counts. BALF protein was
assessed by protein assay (BioRad). Data are presented as s.e.m. of
at least five mice per condition.
[0126] Bleomycin model. Bleomycin experiments were carried out as
described in (Gasse, P., et al. IL-1R1/MyD88 signaling and the
inflammasome are essential in pulmonary inflammation and fibrosis
in mice. J Clin Invest 117, 3786-3799 (2007)). In brief, 6-8 week
old mice were anesthetized and given Bleomycin (0.05 U in 40 .mu.L,
saline) by intranasal instillation. Control mice received an
intranasal instillation of 40 .mu.L saline. Mice were given a daily
intraperitoneal injection of 5 .mu.g Slit or Mock. Control mice
received a daily injection of saline. On day 11, mice were
sacrificed by CO.sub.2 asphyxiation, lungs removed, and homogenized
in 0.5M acetic acid with protease inhibitors (Roche). Homogenates
were incubated overnight at 4.degree. C. with stirring. Samples
were then centrifuged and 1 mL Sircol dye reagent was added to 100
.mu.L of supernatant for 30 minutes. Samples were again
centrifuged, the pellet resuspended with 1 mL alkali reagent, and
analyzed by spectrophotometry (Biocolor). These samples were
compared against a collagen standard curve provided by the
manufacturer. Data are presented as s.e.m. of at least five mice
per condition.
[0127] H5N1 infection. Female 18-20 g BALB/c mice (Charles River
Laboratories) were anesthetized and infected with H5N1 virus
(Influenza A, Duck/MN/1525/81) by intranasal instillation. Mice
were given an intravenous injection of 1.56 .mu.g Slit or Mock
daily for 5 days. Survival rate of mice subjected to H5N1 lung
infection was determined for 21 days with 20 mice per
condition.
[0128] Cecal ligation and puncture (CLP) sepsis model. Seven to
eleven week old male C57BL/6 mice were given an intraperitoneal
injection of 5 .mu.g Slit2N or Mock. One hour later, mice were
anesthetized with isoflurane, and CLP performed as described in
Gomes, R. N., R. T. Figueiredo, F. A. Bozza, P. Pacheco, R. T.
Amancio, A. P. Laranjeira, H. C. Castro-Faria-Neto, P. T. Bozza,
and M. T. Bozza. 2006. Increased susceptibility to septic and
endotoxic shock in monocyte chemoattractant protein 1/cc chemokine
ligand 2-deficient mice correlates with reduced interleukin 10 and
enhanced macrophage migration inhibitory factor production. Shock
26:457-463. Mice continued to receive an intraperitoneal injection
of 5 .mu.g Slit2N or Mock once a day. Mice in the sham operation
group were subjected to identical procedures, except that ligation
and puncture of the cecum were omitted. Survival rate of mice
subjected to CLP was determined for 6 days with N=14 for Slit2N
treatment and N=15 for Mock treatment for Robo4.sup.+/+ mice. For
Robo4.sup.AP/AP mice, N=13 for Slit2N treatment and N=13 for Mock
treatment.
[0129] Evans Blue permeability. Vascular permeability in the lung
was assessed using Evans Blue Albumin (EBA) as described in Moitra
et al. (Moitra, J., S. Sammani, and J. G. Garcia. 2007.
Re-evaluation of Evans Blue dye as a marker of albumin clearance in
murine models of acute lung injury. Transl Res 150:253-265). Five
hours after IT instillation of LPS, 4 hours after CLP, and 3 days
after H5N1 infection, mice were given an IV injection of EBA (20
mg/kg). EBA was allowed to circulate for 1 hour, mice were deeply
anesthetized, and perfused with saline+5 mM EDTA. Lungs were
excised, weighed, and homogenized in 2 mL PBS. Four mL of formamide
(Invitrogen) was added and the samples were incubated overnight at
60.degree. C. to extract Evans Blue dye. The samples were then
centrifuged and supernatants analyzed by spectrophotometry at both
620 and 740 nm. For CLP treated mice, mice were perfused, the
kidneys and spleen were removed, weighed, and placed in formamide
for 48 hours at 60.degree. C. The absorbances were normalized as
described in Moitra et al. and converted to .mu.g Evans Blue dye
per gram wet weight of lungs, kidneys or spleen respectively. Data
are presented as s.e.m. of at least four mice per condition.
[0130] Histology. Twenty-four hours after LPS exposure or CLP, mice
were euthanized by CO.sub.2 asphyxiation. Chest cavities were
opened, and lungs were inflated with ZnSO.sub.2 buffered 10%
Formalin. Formalin fixed tissues were processed routinely, embedded
in paraffin, sectioned at 6 microns, and stained with H&E.
Histologic quantification was modified from methods described in
Gupta et al. (Gupta, N., X. Su, B. Popov, J. W. Lee, V. Serikov,
and M. A. Matthay. 2007. Intrapulmonary delivery of bone
marrow-derived mesenchymal stem cells improves survival and
attenuates endotoxin-induced acute lung injury in mice. J Immunol
179:1855-1863). For H5N1 samples, six days post-infection the right
lobes of the lungs from two animals were harvested and fixed in 10%
neutral buffered formalin. Formalin fixed tissues were processed
routinely, embedded in paraffin, sectioned at 5 microns, stained
with H&E, and evaluated for microscopic lesions by a board
certified veterinary pathologist.
[0131] RNA silencing of Robo4. For each experiment, HMVEC-L, P-4
(Lonza) were grown until 70-80% confluent in EGM-2mv medium (Lonza)
in a 150 cm flask. Prior to trypsinizing, 12 Fibronectin-coated 6.5
mm 3.0 .mu.m pore Transwells (Costar) received 25 .mu.l/well of a
huRobo4siRNA duplex (Qiagen, Hs_ROBO4.sub.--1_HP, #1919431) diluted
to 480 nM in RNA Suspension Buffer (Qiagen), and a second set of 12
transwells received 25 .mu.l/well of an equimolar AllStars
NegativeControl siRNA (Qiagen, #1027280). To form transfection
complexes, siRNAs were premixed for 10 minutes at room temperature
in the Transwells with 25 .mu.l/well of HiPerfect Transfection
Reagent (Qiagen), diluted 1:10 in OptiMEM (Invitrogen). Cells were
lifted, and 150 .mu.l complete medium, containing 2.times.10.sup.4
cells/well were seeded onto the transfection complexes.
Transfectants were incubated for 30 minutes at 37.degree. C., and
800 .mu.l complete medium was added to the lower chamber of each
Transwell. The transfected monolayers were cultured 48 h further,
before performing the In Vitro permeability assay, as described. To
confirm ablation of the Robo4 gene product, two 100 mm dishes were
seeded with the remaining HMVEC-L, at 10.sup.6 cells/dish in 4.5 ml
EGM-2mv, receiving either Robo4si RNA or AllStars Negative Control
transfection complexes consisting of 12.5 .mu.l of 20 .mu.mRNA+70
.mu.l HiPerfect+500 .mu.l Opti MEM, pipetted on top of each dish of
cell suspension. After 30 minutes at 37.degree. C., 6.5 ml complete
media, was added, and the plates were incubated for 48 h, followed
by harvesting of lysate and Western blotting with 1:100 anti-Robo4
(N-17) and 1:200 anti-.beta.Tubullin antibodies (Santa Cruz
Biotechnology).
[0132] Robo4 siRNA knockdown. huRobo4 siRNA duplex
(Hs_ROBO4.sub.--1_HP #1919431, Qiagen) or equimolar AllStars
Negative Control siRNA (#1027280, Qiagen) transfection complexes
were formed according to standard protocol and added to the upper
chamber of Transwell filters. Twenty four hours later, cells were
transfected a second time with huRobo4 or Control siRNA. After an
additional 24 hours, in vitro permeability was assessed as
previously described. Data are presented as mean.+-.s.e.m. of at
least three independent experiments performed in triplicate.
Successful knockdown of Robo4 protein expression was confirmed by
Western Blot using antibodies against Robo4 (N-17) or
.beta.-Tubulin (Santa Cruz Biotechnology).
[0133] In Vitro Permeability Assay. In vitro permeability was
performed as described in Jones et al. (Jones, C. A., N. R. London,
H. Chen, K. W. Park, D. Sauvaget, R. A. Stockton, J. D. Wythe, W.
Suh, F. Larrieu-Lahargue, Y. S. Mukouyama, P. Lindblom, P. Seth, A.
Frias, N. Nishiya, M. H. Ginsberg, H. Gerhardt, K. Zhang, and D. Y.
Li. 2008. Robo4 stabilizes the vascular network by inhibiting
pathologic angiogenesis and endothelial hyperpermeability. Nat Med
14:448-453). The assay was conducted using 100 ng/mL
lipopolysaccharide (serotype 0111:B4, Sigma) for 3 hours, 10 ng/mL
Tumor Necrosis Factor-alpha (TNF-.alpha., R&D Systems) for 6
hours, or 10 ng/mL Interleukin-1 beta (IL-1.beta., R&D Systems)
for 2 hours. As indicated, this was repeated in the presence of 25
.mu.g/mL control rabbit IgG (Jackson ImmunoResearch) or 25 .mu.g/mL
anti-human VE-cadherin antibody (RDI Fitzgerald). Basal
permeability for unstimulated monolayers was set at 100%. Data are
presented as mean.+-.s.e.m of at least three independent
experiments performed in triplicate.
[0134] Subcellular fractionation. HMVEC-lung were treated with
Slit2N or Mock in 0.1% FBS EBM-2 for 1.5 hours. Cells were then
washed twice with ice-cold PBS containing Ca.sup.2+/Mg.sup.2+, once
with HLB buffer (10 mM Tris-HCl PH7.4, 5 mM KCl, 1 mM MgCl.sub.2),
and collected in HLB buffer supplemented with protease inhibitors
(Roche), phosphatase inhibitors (Sigma) and 1 mM DTT. Cells were
then dounce-homogenized (20 strokes). The homogenate was
centrifuged at 400 g for 10 minutes at 4.degree. C. to pellet cell
debris. The resulting supernatant was centrifuged again at 16,000 g
for 30 minutes at 4.degree. C. The pellet was washed once with HLB
and resuspended in RIPA buffer for 30 minutes at 4.degree. C. The
resuspended pellet was centrifuged (16,000 g/15 min at 4.degree.
C.), and the resulting supernatant was saved as soluble membrane
fraction. To obtain the total cell lysate an aliquot was saved
prior to dounce homogenization. RIPA buffer was added to this
aliquot and centrifuged at 13,000 g for 10 minutes at 4.degree. C.
The supernatant was saved and used as total cell lysate. Antibodies
to VE-cadherin were obtained from Cell Signaling, and p120-catenin
and .beta.-catenin from BD biosciences. Densitometry was performed
on at least three independent experiments and data are presented as
mean.+-.s.e.m.
[0135] Immunofluorescence. Immunofluorescence was performed as
described in Jones, C. A., N. R. London, H. Chen, K. W. Park, D.
Sauvaget, R. A. Stockton, J. D. Wythe, W. Suh, F. Larrieu-Lahargue,
Y. S. Mukouyama, P. Lindblom, P. Seth, A. Frias, N. Nishiya, M. H.
Ginsberg, H. Gerhardt, K. Zhang, and D. Y. Li. 2008. Cells were
pre-treated with Slit2N or Mock for 30 minutes followed by
stimulation with 10 ng/mL IL-1.beta. for 3 hours. Primary
antibodies to VE-cadherin (BD biosciences) or p120-catenin (Santa
Cruz) were applied at 4.degree. C. overnight. Images are
representative of three independent experiments.
[0136] Immunoprecipitation. HMVEC-lung were treated with Slit2N or
Mock in 0.1% FBS EBM-2 for 30 minutes. Cells were then stimulated
with 10 ng/mL IL-1.beta. for 10 minutes. HMVEC-lung were then
washed with ice-cold PBS and lysed with ice-cold lysis buffer (10
mM Tris-HCl pH7.4, 50 mM NaCl, 1% NP-40 and 10% glycerol)
supplemented with protease inhibitors, phosphatase inhibitors and 1
mM DTT. Cell lysates were incubated on ice for 30 min and
centrifuged at 13,000 g for 15 minutes to pellet cell debris.
Protein concentrations were determined by BCA assay (PIERCE) and
0.5 mg lysate were incubated with 8 .mu.g of VE-cadherin antibody
(Cell Signaling) and protein A/G sepharose (Santa Cruz) for 1 hour
at 4.degree. C. Complexes were washed three times with lysis
buffer. The immunoprecipitates were subjected to Western blot
analysis. Densitometry was performed on three independent
experiments and data are presented as mean.+-.s.e.m.
[0137] Internalization assay. VE-cadherin internalization was
performed as described Xiao, K., J. Garner, K. M. Buckley, P. A.
Vincent, C. M. Chiasson, E. Dejana, V. Faundez, and A. P.
Kowalczyk. 2005. p120-Catenin regulates clathrin-dependent
endocytosis of VE-cadherin. Mol Biol Cell 16:5141-5151. In brief,
HMVEC-lung were seeded onto chamber slides and cultured for 72
hours. The media was then removed and the cells labeled for 30
minutes at 4.degree. C. with anti-VE-cadherin antibody (clone BV6,
RDI Fitzgerald). Cells were then pre-treated with Slit2N or Mock
for 30 minutes. Excess antibody was removed by washing twice on ice
with ice-cold media. Chamber slides were moved to 37.degree. C. and
incubated for 1 hour with 10 ng/ml IL-1.beta. and 0.6 mM primaquine
in the presence of 10 nM Slit2N or Mock. Cells were acid-washed to
strip the surface-bound VE-cadherin. Monolayers were washed, fixed,
and permeablized. Internalized VE-cadherin antibody was detected
with Alexa 488-conjugated donkey anti-mouse IgG (Molecular Probes).
Images are representative of four independent experiments.
[0138] Lung immunofluorescence. Adult Robo4.sup.+/AP mice were
euthanized by CO.sub.2 asphyxiation. Chest cavities were opened,
lungs inflated with OCT, and frozen quickly in OCT on dry ice. Lung
sections were stained for alkaline phosphatase activity denoting
areas of Robo4 expression as described in Jones, C. A., N. R.
London, H. Chen, K. W. Park, D. Sauvaget, R. A. Stockton, J. D.
Wythe, W. Suh, F. Larrieu-Lahargue, Y. S. Mukouyama, P. Lindblom,
P. Seth, A. Frias, N. Nishiya, M. H. Ginsberg, H. Gerhardt, K.
Zhang, and D. Y. Li. 2008. Robo4 stabilizes the vascular network by
inhibiting pathologic angiogenesis and endothelial
hyperpermeability. Nat Med 14:448-453. Sections were then stained
using primary antibodies against Slit2 (E-20, Santa Cruz) or CD-31
(Pharmingen) followed by fluorescent secondary antibody
staining
[0139] Cytokine/chemokine array. Six hours after CLP, mice were
heavily anesthetized. Whole blood was drawn into ACD (.about.1:9
volume) from the carotid artery. Plasma was isolated by
centrifugation of blood at 4000 g for 10 minutes. Plasma was
analyzed by Quansys Biosciences (Logan, Utah) to quantify cytokine
and chemokine levels. Data are presented as mean.+-.s.e.m. of six
mice per condition. For H5N1 samples, six days after infection
clarified mouse lung homogenates were prepared and inflammatory
cytokine and chemokine profiles determined using mouse cytokine and
chemokine arrays (Quansys Biosciences; Logan, Utah). Data are
presented as mean.+-.s.e.m. of three groups of pooled mice.
[0140] Lung virus titer determination. Performed as described in
Sidwell, R. W., K. W. Bailey, M. H. Wong, D. L. Barnard, and D. F.
Smee. 2005. In vitro and in vivo influenza virus-inhibitory effects
of viramidine. Antiviral Res 68:10-17.
[0141] Lung development. Embryos were dissected, fixed, and
rehydrated as described in Metzger, R. J., O. D. Klein, G. R.
Martin, and M. A. Krasnow. 2008. The branching programme of mouse
lung development. Nature 453:745-750. Lungs were serially
immunostained with anti-PECAM (BD Pharmingen; clone MEC 13.3) and
anti-E-cadherin (clone ECCD-2, Zymed) primary antibodies using a
variation of the method described in Metzger et al.
[0142] Neutrophil migration. HL-60 cells were grown under standard
conditions with RPMI-1640 media supplemented with 10% FBS and 1%
pen/step. Cells induced with 1.2% dimethyl sulphoxide (DMSO) were
obtained by seeding HL-60 cells at 3.times.10.sup.6 per mL in
growth media and culturing for 4 6 days (Collins, S. J., F. W.
Ruscetti, R. E. Gallagher, and R. C. Gallo. 1978. Terminal
differentiation of human promyelocytic leukemia cells induced by
dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci
USA 75:2458-2462). hPMNs were isolated from healthy adult donor
whole blood with ACD using techniques described in Zimmerman, G.
A., T. M. McIntyre, and S. M. Prescott. 1985. Thrombin stimulates
the adherence of neutrophils to human endothelial cells in vitro. J
Clin Invest 76:2235-2246. The leukocyte chemoattractant fMLP (10
.mu.M) along with Slit2 or Mock was placed in the lower wells of a
48-well chemotaxis chamber (Neuroprobe). A fibronectin coated (over
night at 4.degree. C.) polycarbonate membrane (Neuroprobe, 5 .mu.m)
was placed between the chemoattractant and the cells. HL-60 cells
induced with DMSO or hPMNs (50 .mu.l, 50,000 cells) were added to
the upper wells. After incubating at 37.degree. C. for 2 h, cells
on the top surface of the filter were removed and cells that had
migrated through the filter onto the undersurface were fixed and
stained using Diff-Quic stain set (Dade Behring). Migrated cells in
5 high power fields were counted and migration expressed as the
percent of cells migrated compared to cells migrated towards fMLP
in the absence of Slit2 or Mock. Data are presented as s.e.m of at
least three independent experiments.
[0143] Quantitative polymerase chain reaction (qPCR). Total RNA was
extracted from the lungs of saline or LPS treated mice according to
manufacturer s protocol (Nucleospin RNA II kit, Clontech). After
reverse transcription, qPCR was performed with TaqMan assays
(Applied Biosystems) for 18s rRNA and mouse Robo4. For hPMN
studies, RNA was isolated using Trizol (Invitrogen) and qPCR was
performed with TaqMan assays (Applied Biosystems) for human GAPDH
and ROBO1-4.
[0144] Statistical analysis. The Student s t-test, log rank test,
or ANOVA with post-hoc tests, where appropriate, were used to
assess statistical significance. A P value of <0.05 was
considered statistically significant.
[0145] It will be obvious to those having skill in the art that
many changes may be made to the details of the above-described
embodiments without departing from the underlying principles of the
invention. The scope of the present invention should, therefore, be
determined only by the following claims.
Sequence CWU 1
1
1211529PRTHomo sapiens 1Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu
Ser Leu Gly Leu Val1 5 10 15Leu Ala Ile Leu Asn Lys Val Ala Pro Gln
Ala Cys Pro Ala Gln Cys 20 25 30Ser Cys Ser Gly Ser Thr Val Asp Cys
His Gly Leu Ala Leu Arg Ser 35 40 45Val Pro Arg Asn Ile Pro Arg Asn
Thr Glu Arg Leu Asp Leu Asn Gly 50 55 60Asn Asn Ile Thr Arg Ile Thr
Lys Thr Asp Phe Ala Gly Leu Arg His65 70 75 80Leu Arg Val Leu Gln
Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg 85 90 95Gly Ala Phe Gln
Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg 100 105 110Asn His
Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys 115 120
125Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg
130 135 140Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu
Asp Tyr145 150 155 160Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe
Arg Ala Leu Arg Asp 165 170 175Leu Glu Val Leu Thr Leu Asn Asn Asn
Asn Ile Thr Arg Leu Ser Val 180 185 190Ala Ser Phe Asn His Met Pro
Lys Leu Arg Thr Phe Arg Leu His Ser 195 200 205Asn Asn Leu Tyr Cys
Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu 210 215 220Arg Gln Arg
Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser225 230 235
240His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val
245 250 255Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val
Leu His 260 265 270Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val
Asp Cys Arg Gly 275 280 285Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu
Pro Glu Thr Ile Thr Glu 290 295 300Ile Arg Leu Glu Gln Asn Thr Ile
Lys Val Ile Pro Pro Gly Ala Phe305 310 315 320Ser Pro Tyr Lys Lys
Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile 325 330 335Ser Glu Leu
Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser 340 345 350Leu
Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe 355 360
365Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile
370 375 380Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu
Asn Leu385 390 395 400Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile
Ala Lys Gly Thr Phe 405 410 415Ser Pro Leu Arg Ala Ile Gln Thr Met
His Leu Ala Gln Asn Pro Phe 420 425 430Ile Cys Asp Cys His Leu Lys
Trp Leu Ala Asp Tyr Leu His Thr Asn 435 440 445Pro Ile Glu Thr Ser
Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala 450 455 460Asn Lys Arg
Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala465 470 475
480Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu
485 490 495Ser Gly Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys
Arg Cys 500 505 510Glu Gly Thr Thr Val Asp Cys Ser Asn Gln Lys Leu
Asn Lys Ile Pro 515 520 525Glu His Ile Pro Gln Tyr Thr Ala Glu Leu
Arg Leu Asn Asn Asn Glu 530 535 540Phe Thr Val Leu Glu Ala Thr Gly
Ile Phe Lys Lys Leu Pro Gln Leu545 550 555 560Arg Lys Ile Asn Phe
Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly 565 570 575Ala Phe Glu
Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn 580 585 590Arg
Leu Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu 595 600
605Lys Thr Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp
610 615 620Ser Phe Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr
Asp Asn625 630 635 640Gln Ile Thr Thr Val Ala Pro Gly Ala Phe Asp
Thr Leu His Ser Leu 645 650 655Ser Thr Leu Asn Leu Leu Ala Asn Pro
Phe Asn Cys Asn Cys Tyr Leu 660 665 670Ala Trp Leu Gly Glu Trp Leu
Arg Lys Lys Arg Ile Val Thr Gly Asn 675 680 685Pro Arg Cys Gln Lys
Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp 690 695 700Val Ala Ile
Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser705 710 715
720Cys Ser Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr
725 730 735Val Val Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys
Gly Ile 740 745 750Pro Arg Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn
Gln Phe Thr Leu 755 760 765Val Pro Lys Glu Leu Ser Asn Tyr Lys His
Leu Thr Leu Ile Asp Leu 770 775 780Ser Asn Asn Arg Ile Ser Thr Leu
Ser Asn Gln Ser Phe Ser Asn Met785 790 795 800Thr Gln Leu Leu Thr
Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile 805 810 815Pro Pro Arg
Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu 820 825 830His
Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu 835 840
845Ser Ala Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp
850 855 860Cys Asn Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr
Lys Glu865 870 875 880Pro Gly Ile Ala Arg Cys Ala Gly Pro Gly Glu
Met Ala Asp Lys Leu 885 890 895Leu Leu Thr Thr Pro Ser Lys Lys Phe
Thr Cys Gln Gly Pro Val Asp 900 905 910Val Asn Ile Leu Ala Lys Cys
Asn Pro Cys Leu Ser Asn Pro Cys Lys 915 920 925Asn Asp Gly Thr Cys
Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr 930 935 940Cys Pro Tyr
Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala945 950 955
960Cys Ile Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu
965 970 975Gly Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe
Glu Gly 980 985 990Glu Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp
Asn Asp Cys Glu 995 1000 1005Asn Asn Ser Thr Cys Val Asp Gly Ile
Asn Asn Tyr Thr Cys Leu 1010 1015 1020Cys Pro Pro Glu Tyr Thr Gly
Glu Leu Cys Glu Glu Lys Leu Asp 1025 1030 1035Phe Cys Ala Gln Asp
Leu Asn Pro Cys Gln His Asp Ser Lys Cys 1040 1045 1050Ile Leu Thr
Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr 1055 1060 1065Val
Gly Glu His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn 1070 1075
1080Lys Cys Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr
1085 1090 1095Thr Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys
Glu Phe 1100 1105 1110Ser Pro Pro Met Val Leu Pro Arg Thr Ser Pro
Cys Asp Asn Phe 1115 1120 1125Asp Cys Gln Asn Gly Ala Gln Cys Ile
Val Arg Ile Asn Glu Pro 1130 1135 1140Ile Cys Gln Cys Leu Pro Gly
Tyr Gln Gly Glu Lys Cys Glu Lys 1145 1150 1155Leu Val Ser Val Asn
Phe Ile Asn Lys Glu Ser Tyr Leu Gln Ile 1160 1165 1170Pro Ser Ala
Lys Val Arg Pro Gln Thr Asn Ile Thr Leu Gln Ile 1175 1180 1185Ala
Thr Asp Glu Asp Ser Gly Ile Leu Leu Tyr Lys Gly Asp Lys 1190 1195
1200Asp His Ile Ala Val Glu Leu Tyr Arg Gly Arg Val Arg Ala Ser
1205 1210 1215Tyr Asp Thr Gly Ser His Pro Ala Ser Ala Ile Tyr Ser
Val Glu 1220 1225 1230Thr Ile Asn Asp Gly Asn Phe His Ile Val Glu
Leu Leu Ala Leu 1235 1240 1245Asp Gln Ser Leu Ser Leu Ser Val Asp
Gly Gly Asn Pro Lys Ile 1250 1255 1260Ile Thr Asn Leu Ser Lys Gln
Ser Thr Leu Asn Phe Asp Ser Pro 1265 1270 1275Leu Tyr Val Gly Gly
Met Pro Gly Lys Ser Asn Val Ala Ser Leu 1280 1285 1290Arg Gln Ala
Pro Gly Gln Asn Gly Thr Ser Phe His Gly Cys Ile 1295 1300 1305Arg
Asn Leu Tyr Ile Asn Ser Glu Leu Gln Asp Phe Gln Lys Val 1310 1315
1320Pro Met Gln Thr Gly Ile Leu Pro Gly Cys Glu Pro Cys His Lys
1325 1330 1335Lys Val Cys Ala His Gly Thr Cys Gln Pro Ser Ser Gln
Ala Gly 1340 1345 1350Phe Thr Cys Glu Cys Gln Glu Gly Trp Met Gly
Pro Leu Cys Asp 1355 1360 1365Gln Arg Thr Asn Asp Pro Cys Leu Gly
Asn Lys Cys Val His Gly 1370 1375 1380Thr Cys Leu Pro Ile Asn Ala
Phe Ser Tyr Ser Cys Lys Cys Leu 1385 1390 1395Glu Gly His Gly Gly
Val Leu Cys Asp Glu Glu Glu Asp Leu Phe 1400 1405 1410Asn Pro Cys
Gln Ala Ile Lys Cys Lys His Gly Lys Cys Arg Leu 1415 1420 1425Ser
Gly Leu Gly Gln Pro Tyr Cys Glu Cys Ser Ser Gly Tyr Thr 1430 1435
1440Gly Asp Ser Cys Asp Arg Glu Ile Ser Cys Arg Gly Glu Arg Ile
1445 1450 1455Arg Asp Tyr Tyr Gln Lys Gln Gln Gly Tyr Ala Ala Cys
Gln Thr 1460 1465 1470Thr Lys Lys Val Ser Arg Leu Glu Cys Arg Gly
Gly Cys Ala Gly 1475 1480 1485Gly Gln Cys Cys Gly Pro Leu Arg Ser
Lys Arg Arg Lys Tyr Ser 1490 1495 1500Phe Glu Cys Thr Asp Gly Ser
Ser Phe Val Asp Glu Val Glu Lys 1505 1510 1515Val Val Lys Cys Gly
Cys Thr Arg Cys Val Ser 1520 152521119PRTHomo sapiens 2Met Arg Gly
Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val1 5 10 15Leu Ala
Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys 20 25 30Ser
Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser 35 40
45Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly
50 55 60Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg
His65 70 75 80Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr
Ile Glu Arg 85 90 95Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu
Arg Leu Asn Arg 100 105 110Asn His Leu Gln Leu Phe Pro Glu Leu Leu
Phe Leu Gly Thr Ala Lys 115 120 125Leu Tyr Arg Leu Asp Leu Ser Glu
Asn Gln Ile Gln Ala Ile Pro Arg 130 135 140Lys Ala Phe Arg Gly Ala
Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr145 150 155 160Asn Gln Ile
Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp 165 170 175Leu
Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val 180 185
190Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser
195 200 205Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp
Trp Leu 210 215 220Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys
Met Gly Pro Ser225 230 235 240His Leu Arg Gly His Asn Val Ala Glu
Val Gln Lys Arg Glu Phe Val 245 250 255Cys Ser Gly His Gln Ser Phe
Met Ala Pro Ser Cys Ser Val Leu His 260 265 270Cys Pro Ala Ala Cys
Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly 275 280 285Lys Gly Leu
Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu 290 295 300Ile
Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe305 310
315 320Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln
Ile 325 330 335Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser
Leu Asn Ser 340 345 350Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu
Pro Lys Ser Leu Phe 355 360 365Glu Gly Leu Phe Ser Leu Gln Leu Leu
Leu Leu Asn Ala Asn Lys Ile 370 375 380Asn Cys Leu Arg Val Asp Ala
Phe Gln Asp Leu His Asn Leu Asn Leu385 390 395 400Leu Ser Leu Tyr
Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe 405 410 415Ser Pro
Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe 420 425
430Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn
435 440 445Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg
Leu Ala 450 455 460Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe
Arg Cys Ser Ala465 470 475 480Lys Glu Gln Tyr Phe Ile Pro Gly Thr
Glu Asp Tyr Arg Ser Lys Leu 485 490 495Ser Gly Asp Cys Phe Ala Asp
Leu Ala Cys Pro Glu Lys Cys Arg Cys 500 505 510Glu Gly Thr Thr Val
Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro 515 520 525Glu His Ile
Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu 530 535 540Phe
Thr Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu545 550
555 560Arg Lys Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu
Gly 565 570 575Ala Phe Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu
Thr Ser Asn 580 585 590Arg Leu Glu Asn Val Gln His Lys Met Phe Lys
Gly Leu Glu Ser Leu 595 600 605Lys Thr Leu Met Leu Arg Ser Asn Arg
Ile Thr Cys Val Gly Asn Asp 610 615 620Ser Phe Ile Gly Leu Ser Ser
Val Arg Leu Leu Ser Leu Tyr Asp Asn625 630 635 640Gln Ile Thr Thr
Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu 645 650 655Ser Thr
Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu 660 665
670Ala Trp Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn
675 680 685Pro Arg Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile
Gln Asp 690 695 700Val Ala Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn
Asp Asp Asn Ser705 710 715 720Cys Ser Pro Leu Ser Arg Cys Pro Thr
Glu Cys Thr Cys Leu Asp Thr 725 730 735Val Val Arg Cys Ser Asn Lys
Gly Leu Lys Val Leu Pro Lys Gly Ile 740 745 750Pro Arg Asp Val Thr
Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu 755 760 765Val Pro Lys
Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu 770 775 780Ser
Asn Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Asn Met Thr785 790
795 800Gln Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile
Pro 805 810 815Pro Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu
Ser Leu His 820 825 830Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala
Phe Asn Asp Leu Ser 835 840 845Ala Leu Ser His Leu Ala Ile Gly Ala
Asn Pro Leu Tyr Cys Asp Cys 850 855 860Asn Met Gln Trp Leu Ser Asp
Trp Val Lys Ser Glu Tyr Lys Glu Pro865 870 875 880Gly Ile Ala Arg
Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu 885 890 895Leu Thr
Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val 900 905
910Asn Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn
915 920 925Asp Gly Thr Cys Asn Ser Asp
Pro Val Asp Phe Tyr Arg Cys Thr Cys 930 935 940Pro Tyr Gly Phe Lys
Gly Gln Asp Cys Asp Val Pro Ile His Ala Cys945 950 955 960Ile Ser
Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu Gly 965 970
975Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu
980 985 990Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys
Glu Asn 995 1000 1005Asn Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr
Thr Cys Leu Cys 1010 1015 1020Pro Pro Glu Tyr Thr Gly Glu Leu Cys
Glu Glu Lys Leu Asp Phe 1025 1030 1035Cys Ala Gln Asp Leu Asn Pro
Cys Gln His Asp Ser Lys Cys Ile 1040 1045 1050Leu Thr Pro Lys Gly
Phe Lys Cys Asp Cys Thr Pro Gly Tyr Val 1055 1060 1065Gly Glu His
Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn Lys 1070 1075 1080Cys
Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr Thr 1085 1090
1095Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe Ser
1100 1105 1110Pro Pro Met Val Leu Pro 11153234PRTHomo sapiens 3Leu
His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys1 5 10
15Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile
20 25 30Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro
Gly 35 40 45Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser
Asn Asn 50 55 60Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu
Arg Ser Leu65 70 75 80Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr
Glu Leu Pro Lys Ser 85 90 95Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu
Leu Leu Leu Asn Ala Asn 100 105 110Lys Ile Asn Cys Leu Arg Val Asp
Ala Phe Gln Asp Leu His Asn Leu 115 120 125Asn Leu Leu Ser Leu Tyr
Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly 130 135 140Thr Phe Ser Pro
Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn145 150 155 160Pro
Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His 165 170
175Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg
180 185 190Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe
Arg Cys 195 200 205Ser Ala Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu
Asp Tyr Arg Ser 210 215 220Lys Leu Ser Gly Asp Cys Phe Ala Asp
Leu225 23041093PRTHomo sapiens 4Gln Ala Cys Pro Ala Gln Cys Ser Cys
Ser Gly Ser Thr Val Asp Cys1 5 10 15His Gly Leu Ala Leu Arg Ser Val
Pro Arg Asn Ile Pro Arg Asn Thr 20 25 30Glu Arg Leu Asp Leu Asn Gly
Asn Asn Ile Thr Arg Ile Thr Lys Thr 35 40 45Asp Phe Ala Gly Leu Arg
His Leu Arg Val Leu Gln Leu Met Glu Asn 50 55 60Lys Ile Ser Thr Ile
Glu Arg Gly Ala Phe Gln Asp Leu Lys Glu Leu65 70 75 80Glu Arg Leu
Arg Leu Asn Arg Asn His Leu Gln Leu Phe Pro Glu Leu 85 90 95Leu Phe
Leu Gly Thr Ala Lys Leu Tyr Arg Leu Asp Leu Ser Glu Asn 100 105
110Gln Ile Gln Ala Ile Pro Arg Lys Ala Phe Arg Gly Ala Val Asp Ile
115 120 125Lys Asn Leu Gln Leu Asp Tyr Asn Gln Ile Ser Cys Ile Glu
Asp Gly 130 135 140Ala Phe Arg Ala Leu Arg Asp Leu Glu Val Leu Thr
Leu Asn Asn Asn145 150 155 160Asn Ile Thr Arg Leu Ser Val Ala Ser
Phe Asn His Met Pro Lys Leu 165 170 175Arg Thr Phe Arg Leu His Ser
Asn Asn Leu Tyr Cys Asp Cys His Leu 180 185 190Ala Trp Leu Ser Asp
Trp Leu Arg Gln Arg Pro Arg Val Gly Leu Tyr 195 200 205Thr Gln Cys
Met Gly Pro Ser His Leu Arg Gly His Asn Val Ala Glu 210 215 220Val
Gln Lys Arg Glu Phe Val Cys Ser Asp Glu Glu Glu Gly His Gln225 230
235 240Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro Ala Ala
Cys 245 250 255Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly
Leu Thr Glu 260 265 270Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu
Ile Arg Leu Glu Gln 275 280 285Asn Thr Ile Lys Val Ile Pro Pro Gly
Ala Phe Ser Pro Tyr Lys Lys 290 295 300Leu Arg Arg Ile Asp Leu Ser
Asn Asn Gln Ile Ser Glu Leu Ala Pro305 310 315 320Asp Ala Phe Gln
Gly Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly 325 330 335Asn Lys
Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly Leu Phe Ser 340 345
350Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val
355 360 365Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser Leu
Tyr Asp 370 375 380Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser
Pro Leu Arg Ala385 390 395 400Ile Gln Thr Met His Leu Ala Gln Asn
Pro Phe Ile Cys Asp Cys His 405 410 415Leu Lys Trp Leu Ala Asp Tyr
Leu His Thr Asn Pro Ile Glu Thr Ser 420 425 430Gly Ala Arg Cys Thr
Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly 435 440 445Gln Ile Lys
Ser Lys Lys Phe Arg Cys Ser Gly Thr Glu Asp Tyr Arg 450 455 460Ser
Lys Leu Ser Gly Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys465 470
475 480Cys Arg Cys Glu Gly Thr Thr Val Asp Cys Ser Asn Gln Lys Leu
Asn 485 490 495Lys Ile Pro Glu His Ile Pro Gln Tyr Thr Ala Glu Leu
Arg Leu Asn 500 505 510Asn Asn Glu Phe Thr Val Leu Glu Ala Thr Gly
Ile Phe Lys Lys Leu 515 520 525Pro Gln Leu Arg Lys Ile Asn Phe Ser
Asn Asn Lys Ile Thr Asp Ile 530 535 540Glu Glu Gly Ala Phe Glu Gly
Ala Ser Gly Val Asn Glu Ile Leu Leu545 550 555 560Thr Ser Asn Arg
Leu Glu Asn Val Gln His Lys Met Phe Lys Gly Leu 565 570 575Glu Ser
Leu Lys Thr Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val 580 585
590Gly Asn Asp Ser Phe Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu
595 600 605Tyr Asp Asn Gln Ile Thr Thr Val Ala Pro Gly Ala Phe Asp
Thr Leu 610 615 620His Ser Leu Ser Thr Leu Asn Leu Leu Ala Asn Pro
Phe Asn Cys Asn625 630 635 640Cys Tyr Leu Ala Trp Leu Gly Glu Trp
Leu Arg Lys Lys Arg Ile Val 645 650 655Thr Gly Asn Pro Arg Cys Gln
Lys Pro Tyr Phe Leu Lys Glu Ile Pro 660 665 670Ile Gln Asp Val Ala
Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp 675 680 685Asp Asn Ser
Cys Ser Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys 690 695 700Leu
Asp Thr Val Val Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro705 710
715 720Lys Gly Ile Pro Arg Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn
Gln 725 730 735Phe Thr Leu Val Pro Lys Glu Leu Ser Asn Tyr Lys His
Leu Thr Leu 740 745 750Ile Asp Leu Ser Asn Asn Arg Ile Ser Thr Leu
Ser Asn Gln Ser Phe 755 760 765Ser Asn Met Thr Gln Leu Leu Thr Leu
Ile Leu Ser Tyr Asn Arg Leu 770 775 780Arg Cys Ile Pro Pro Arg Thr
Phe Asp Gly Leu Lys Ser Leu Arg Leu785 790 795 800Leu Ser Leu His
Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe 805 810 815Asn Asp
Leu Ser Ala Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu 820 825
830Tyr Cys Asp Cys Asn Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu
835 840 845Tyr Lys Glu Pro Gly Ile Ala Arg Cys Ala Gly Pro Gly Glu
Met Ala 850 855 860Asp Lys Leu Leu Leu Thr Thr Pro Ser Lys Lys Phe
Thr Cys Gln Gly865 870 875 880Pro Val Asp Val Asn Ile Leu Ala Lys
Cys Asn Pro Cys Leu Ser Asn 885 890 895Pro Cys Lys Asn Asp Gly Thr
Cys Asn Ser Asp Pro Val Asp Phe Tyr 900 905 910Arg Cys Thr Cys Pro
Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro 915 920 925Ile His Ala
Cys Ile Ser Asn Pro Cys Lys His Gly Gly Thr Cys His 930 935 940Leu
Lys Glu Gly Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly945 950
955 960Phe Glu Gly Glu Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp
Asn 965 970 975Asp Cys Glu Asn Asn Ser Thr Cys Val Asp Gly Ile Asn
Asn Tyr Thr 980 985 990Cys Leu Cys Pro Pro Glu Tyr Thr Gly Glu Leu
Cys Glu Glu Lys Leu 995 1000 1005Asp Phe Cys Ala Gln Asp Leu Asn
Pro Cys Gln His Asp Ser Lys 1010 1015 1020Cys Ile Leu Thr Pro Lys
Gly Phe Lys Cys Asp Cys Thr Pro Gly 1025 1030 1035Tyr Val Gly Glu
His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp 1040 1045 1050Asn Lys
Cys Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly 1055 1060
1065Tyr Thr Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu
1070 1075 1080Phe Ser Pro Pro Met Val Leu Pro Arg Thr 1085
109051507PRTHomo sapiens 5Ile Leu Asn Lys Val Ala Pro Gln Ala Cys
Pro Ala Gln Cys Ser Cys1 5 10 15Ser Gly Ser Thr Val Asp Cys His Gly
Leu Ala Leu Arg Ser Val Pro 20 25 30Arg Asn Ile Pro Arg Asn Thr Glu
Arg Leu Asp Leu Asn Gly Asn Asn 35 40 45Ile Thr Arg Ile Thr Lys Thr
Asp Phe Ala Gly Leu Arg His Leu Arg 50 55 60Val Leu Gln Leu Met Glu
Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala65 70 75 80Phe Gln Asp Leu
Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His 85 90 95Leu Gln Leu
Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr 100 105 110Arg
Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala 115 120
125Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln
130 135 140Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp
Leu Glu145 150 155 160Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg
Leu Ser Val Ala Ser 165 170 175Phe Asn His Met Pro Lys Leu Arg Thr
Phe Arg Leu His Ser Asn Asn 180 185 190Leu Tyr Cys Asp Cys His Leu
Ala Trp Leu Ser Asp Trp Leu Arg Gln 195 200 205Arg Pro Arg Val Gly
Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu 210 215 220Arg Gly His
Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser225 230 235
240Asp Glu Glu Glu Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val
245 250 255Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val
Asp Cys 260 265 270Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu
Pro Glu Thr Ile 275 280 285Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile
Lys Val Ile Pro Pro Gly 290 295 300Ala Phe Ser Pro Tyr Lys Lys Leu
Arg Arg Ile Asp Leu Ser Asn Asn305 310 315 320Gln Ile Ser Glu Leu
Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu 325 330 335Asn Ser Leu
Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser 340 345 350Leu
Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn 355 360
365Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu
370 375 380Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala
Lys Gly385 390 395 400Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met
His Leu Ala Gln Asn 405 410 415Pro Phe Ile Cys Asp Cys His Leu Lys
Trp Leu Ala Asp Tyr Leu His 420 425 430Thr Asn Pro Ile Glu Thr Ser
Gly Ala Arg Cys Thr Ser Pro Arg Arg 435 440 445Leu Ala Asn Lys Arg
Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys 450 455 460Ser Gly Thr
Glu Asp Tyr Arg Ser Lys Leu Ser Gly Asp Cys Phe Ala465 470 475
480Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly Thr Thr Val Asp
485 490 495Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His Ile Pro
Gln Tyr 500 505 510Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr
Val Leu Glu Ala 515 520 525Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu
Arg Lys Ile Asn Phe Ser 530 535 540Asn Asn Lys Ile Thr Asp Ile Glu
Glu Gly Ala Phe Glu Gly Ala Ser545 550 555 560Gly Val Asn Glu Ile
Leu Leu Thr Ser Asn Arg Leu Glu Asn Val Gln 565 570 575His Lys Met
Phe Lys Gly Leu Glu Ser Leu Lys Thr Leu Met Leu Arg 580 585 590Ser
Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe Ile Gly Leu Ser 595 600
605Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala
610 615 620Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn
Leu Leu625 630 635 640Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala
Trp Leu Gly Glu Trp 645 650 655Leu Arg Lys Lys Arg Ile Val Thr Gly
Asn Pro Arg Cys Gln Lys Pro 660 665 670Tyr Phe Leu Lys Glu Ile Pro
Ile Gln Asp Val Ala Ile Gln Asp Phe 675 680 685Thr Cys Asp Asp Gly
Asn Asp Asp Asn Ser Cys Ser Pro Leu Ser Arg 690 695 700Cys Pro Thr
Glu Cys Thr Cys Leu Asp Thr Val Val Arg Cys Ser Asn705 710 715
720Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg Asp Val Thr Glu
725 730 735Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro Lys Glu
Leu Ser 740 745 750Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn
Asn Arg Ile Ser 755 760 765Thr Leu Ser Asn Gln Ser Phe Ser Asn Met
Thr Gln Leu Leu Thr Leu 770 775 780Ile Leu Ser Tyr Asn Arg Leu Arg
Cys Ile Pro Pro Arg Thr Phe Asp785 790 795 800Gly Leu Lys Ser Leu
Arg Leu Leu Ser Leu His Gly Asn Asp Ile Ser 805 810 815Val Val Pro
Glu Gly Ala Phe Asn Asp Leu Ser Ala Leu Ser His Leu 820 825 830Ala
Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn Met Gln Trp Leu 835 840
845Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly Ile Ala Arg Cys
850 855 860Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu Thr Thr
Pro Ser865 870 875 880Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val
Asn Ile Leu Ala Lys 885 890 895Cys Asn Pro Cys Leu Ser Asn Pro Cys
Lys Asn Asp Gly Thr Cys Asn 900 905 910Ser Asp Pro Val Asp Phe Tyr
Arg Cys Thr Cys Pro Tyr Gly Phe Lys 915 920 925Gly Gln Asp Cys Asp
Val Pro Ile His Ala Cys Ile Ser Asn Pro Cys 930 935 940Lys His Gly
Gly Thr Cys His Leu Lys Glu Gly Glu Glu Asp Gly Phe945
950 955 960Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn Cys Glu
Val Asn 965 970 975Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn
Ser Thr Cys Val 980 985 990Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys
Pro Pro Glu Tyr Thr Gly 995 1000 1005Glu Leu Cys Glu Glu Lys Leu
Asp Phe Cys Ala Gln Asp Leu Asn 1010 1015 1020Pro Cys Gln His Asp
Ser Lys Cys Ile Leu Thr Pro Lys Gly Phe 1025 1030 1035Lys Cys Asp
Cys Thr Pro Gly Tyr Val Gly Glu His Cys Asp Ile 1040 1045 1050Asp
Phe Asp Asp Cys Gln Asp Asn Lys Cys Lys Asn Gly Ala His 1055 1060
1065Cys Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile Cys Pro Glu Gly
1070 1075 1080Tyr Ser Gly Leu Phe Cys Glu Phe Ser Pro Pro Met Val
Leu Pro 1085 1090 1095Arg Thr Ser Pro Cys Asp Asn Phe Asp Cys Gln
Asn Gly Ala Gln 1100 1105 1110Cys Ile Val Arg Ile Asn Glu Pro Ile
Cys Gln Cys Leu Pro Gly 1115 1120 1125Tyr Gln Gly Glu Lys Cys Glu
Lys Leu Val Ser Val Asn Phe Ile 1130 1135 1140Asn Lys Glu Ser Tyr
Leu Gln Ile Pro Ser Ala Lys Val Arg Pro 1145 1150 1155Gln Thr Asn
Ile Thr Leu Gln Ile Ala Thr Asp Glu Asp Ser Gly 1160 1165 1170Ile
Leu Leu Tyr Lys Gly Asp Lys Asp His Ile Ala Val Glu Leu 1175 1180
1185Tyr Arg Gly Arg Val Arg Ala Ser Tyr Asp Thr Gly Ser His Pro
1190 1195 1200Ala Ser Ala Ile Tyr Ser Val Glu Thr Ile Asn Asp Gly
Asn Phe 1205 1210 1215His Ile Val Glu Leu Leu Ala Leu Asp Gln Ser
Leu Ser Leu Ser 1220 1225 1230Val Asp Gly Gly Asn Pro Lys Ile Ile
Thr Asn Leu Ser Lys Gln 1235 1240 1245Ser Thr Leu Asn Phe Asp Ser
Pro Leu Tyr Val Gly Gly Met Pro 1250 1255 1260Gly Lys Ser Asn Val
Ala Ser Leu Arg Gln Ala Pro Gly Gln Asn 1265 1270 1275Gly Thr Ser
Phe His Gly Cys Ile Arg Asn Leu Tyr Ile Asn Ser 1280 1285 1290Glu
Leu Gln Asp Phe Gln Lys Val Pro Met Gln Thr Gly Ile Leu 1295 1300
1305Pro Gly Cys Glu Pro Cys His Lys Lys Val Cys Ala His Gly Thr
1310 1315 1320Cys Gln Pro Ser Ser Gln Ala Gly Phe Thr Cys Glu Cys
Gln Glu 1325 1330 1335Gly Trp Met Gly Pro Leu Cys Asp Gln Arg Thr
Asn Asp Pro Cys 1340 1345 1350Leu Gly Asn Lys Cys Val His Gly Thr
Cys Leu Pro Ile Asn Ala 1355 1360 1365Phe Ser Tyr Ser Cys Lys Cys
Leu Glu Gly His Gly Gly Val Leu 1370 1375 1380Cys Asp Glu Glu Glu
Asp Leu Phe Asn Pro Cys Gln Ala Ile Lys 1385 1390 1395Cys Lys His
Gly Lys Cys Arg Leu Ser Gly Leu Gly Gln Pro Tyr 1400 1405 1410Cys
Glu Cys Ser Ser Gly Tyr Thr Gly Asp Ser Cys Asp Arg Glu 1415 1420
1425Ile Ser Cys Arg Gly Glu Arg Ile Arg Asp Tyr Tyr Gln Lys Gln
1430 1435 1440Gln Gly Tyr Ala Ala Cys Gln Thr Thr Lys Lys Val Ser
Arg Leu 1445 1450 1455Glu Cys Arg Gly Gly Cys Ala Gly Gly Gln Cys
Cys Gly Pro Leu 1460 1465 1470Arg Ser Lys Arg Arg Lys Tyr Ser Phe
Glu Cys Thr Asp Gly Ser 1475 1480 1485Ser Phe Val Asp Glu Val Glu
Lys Val Val Lys Cys Gly Cys Thr 1490 1495 1500Arg Cys Val Ser
150561501PRTHomo sapiens 6Ile Leu Asn Lys Val Ala Pro Gln Ala Cys
Pro Ala Gln Cys Ser Cys1 5 10 15Ser Gly Ser Thr Val Asp Cys His Gly
Leu Ala Leu Arg Ser Val Pro 20 25 30Arg Asn Ile Pro Arg Asn Thr Glu
Arg Leu Asp Leu Asn Gly Asn Asn 35 40 45Ile Thr Arg Ile Thr Lys Thr
Asp Phe Ala Gly Leu Arg His Leu Arg 50 55 60Val Leu Gln Leu Met Glu
Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala65 70 75 80Phe Gln Asp Leu
Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His 85 90 95Leu Gln Leu
Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr 100 105 110Arg
Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala 115 120
125Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln
130 135 140Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp
Leu Glu145 150 155 160Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg
Leu Ser Val Ala Ser 165 170 175Phe Asn His Met Pro Lys Leu Arg Thr
Phe Arg Leu His Ser Asn Asn 180 185 190Leu Tyr Cys Asp Cys His Leu
Ala Trp Leu Ser Asp Trp Leu Arg Gln 195 200 205Arg Pro Arg Val Gly
Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu 210 215 220Arg Gly His
Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser225 230 235
240Asp Glu Glu Glu Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val
245 250 255Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val
Asp Cys 260 265 270Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu
Pro Glu Thr Ile 275 280 285Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile
Lys Val Ile Pro Pro Gly 290 295 300Ala Phe Ser Pro Tyr Lys Lys Leu
Arg Arg Ile Asp Leu Ser Asn Asn305 310 315 320Gln Ile Ser Glu Leu
Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu 325 330 335Asn Ser Leu
Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser 340 345 350Leu
Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn 355 360
365Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu
370 375 380Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala
Lys Gly385 390 395 400Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met
His Leu Ala Gln Asn 405 410 415Pro Phe Ile Cys Asp Cys His Leu Lys
Trp Leu Ala Asp Tyr Leu His 420 425 430Thr Asn Pro Ile Glu Thr Ser
Gly Ala Arg Cys Thr Ser Pro Arg Arg 435 440 445Leu Ala Asn Lys Arg
Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys 450 455 460Ser Gly Thr
Glu Asp Tyr Arg Ser Lys Leu Ser Gly Asp Cys Phe Ala465 470 475
480Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly Thr Thr Val Asp
485 490 495Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His Ile Pro
Gln Tyr 500 505 510Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr
Val Leu Glu Ala 515 520 525Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu
Arg Lys Ile Asn Phe Ser 530 535 540Asn Asn Lys Ile Thr Asp Ile Glu
Glu Gly Ala Phe Glu Gly Ala Ser545 550 555 560Gly Val Asn Glu Ile
Leu Leu Thr Ser Asn Arg Leu Glu Asn Val Gln 565 570 575His Lys Met
Phe Lys Gly Leu Glu Ser Leu Lys Thr Leu Met Leu Arg 580 585 590Ser
Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe Ile Gly Leu Ser 595 600
605Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala
610 615 620Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn
Leu Leu625 630 635 640Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala
Trp Leu Gly Glu Trp 645 650 655Leu Arg Lys Lys Arg Ile Val Thr Gly
Asn Pro Arg Cys Gln Lys Pro 660 665 670Tyr Phe Leu Lys Glu Ile Pro
Ile Gln Asp Val Ala Ile Gln Asp Phe 675 680 685Thr Cys Asp Asp Gly
Asn Asp Asp Asn Ser Cys Ser Pro Leu Ser Arg 690 695 700Cys Pro Thr
Glu Cys Thr Cys Leu Asp Thr Val Val Arg Cys Ser Asn705 710 715
720Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg Asp Val Thr Glu
725 730 735Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro Lys Glu
Leu Ser 740 745 750Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn
Asn Arg Ile Ser 755 760 765Thr Leu Ser Asn Gln Ser Phe Ser Asn Met
Thr Gln Leu Leu Thr Leu 770 775 780Ile Leu Ser Tyr Asn Arg Leu Arg
Cys Ile Pro Pro Arg Thr Phe Asp785 790 795 800Gly Leu Lys Ser Leu
Arg Leu Leu Ser Leu His Gly Asn Asp Ile Ser 805 810 815Val Val Pro
Glu Gly Ala Phe Asn Asp Leu Ser Ala Leu Ser His Leu 820 825 830Ala
Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn Met Gln Trp Leu 835 840
845Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly Ile Ala Arg Cys
850 855 860Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu Thr Thr
Pro Ser865 870 875 880Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val
Asn Ile Leu Ala Lys 885 890 895Cys Asn Pro Cys Leu Ser Asn Pro Cys
Lys Asn Asp Gly Thr Cys Asn 900 905 910Ser Asp Pro Val Asp Phe Tyr
Arg Cys Thr Cys Pro Tyr Gly Phe Lys 915 920 925Gly Gln Asp Cys Asp
Val Pro Ile His Ala Cys Ile Ser Asn Pro Cys 930 935 940Lys His Gly
Gly Thr Cys His Leu Lys Glu Gly Glu Glu Asp Gly Phe945 950 955
960Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn Cys Glu Val Asn
965 970 975Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn Ser Thr
Cys Val 980 985 990Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro
Glu Tyr Thr Gly 995 1000 1005Glu Leu Cys Glu Glu Lys Leu Asp Phe
Cys Ala Gln Asp Leu Asn 1010 1015 1020Pro Cys Gln His Asp Ser Lys
Cys Ile Leu Thr Pro Lys Gly Phe 1025 1030 1035Lys Cys Asp Cys Thr
Pro Gly Tyr Val Gly Glu His Cys Asp Ile 1040 1045 1050Asp Phe Asp
Asp Cys Gln Asp Asn Lys Cys Lys Asn Gly Ala His 1055 1060 1065Cys
Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile Cys Pro Glu Gly 1070 1075
1080Tyr Ser Gly Leu Phe Cys Glu Phe Ser Arg Thr Ser Pro Cys Asp
1085 1090 1095Asn Phe Asp Cys Gln Asn Gly Ala Gln Cys Ile Val Arg
Ile Asn 1100 1105 1110Glu Pro Ile Cys Gln Cys Leu Pro Gly Tyr Gln
Gly Glu Lys Cys 1115 1120 1125Glu Lys Leu Val Ser Val Asn Phe Ile
Asn Lys Glu Ser Tyr Leu 1130 1135 1140Gln Ile Pro Ser Ala Lys Val
Arg Pro Gln Thr Asn Ile Thr Leu 1145 1150 1155Gln Ile Ala Thr Asp
Glu Asp Ser Gly Ile Leu Leu Tyr Lys Gly 1160 1165 1170Asp Lys Asp
His Ile Ala Val Glu Leu Tyr Arg Gly Arg Val Arg 1175 1180 1185Ala
Ser Tyr Asp Thr Gly Ser His Pro Ala Ser Ala Ile Tyr Ser 1190 1195
1200Val Glu Thr Ile Asn Asp Gly Asn Phe His Ile Val Glu Leu Leu
1205 1210 1215Ala Leu Asp Gln Ser Leu Ser Leu Ser Val Asp Gly Gly
Asn Pro 1220 1225 1230Lys Ile Ile Thr Asn Leu Ser Lys Gln Ser Thr
Leu Asn Phe Asp 1235 1240 1245Ser Pro Leu Tyr Val Gly Gly Met Pro
Gly Lys Ser Asn Val Ala 1250 1255 1260Ser Leu Arg Gln Ala Pro Gly
Gln Asn Gly Thr Ser Phe His Gly 1265 1270 1275Cys Ile Arg Asn Leu
Tyr Ile Asn Ser Glu Leu Gln Asp Phe Gln 1280 1285 1290Lys Val Pro
Met Gln Thr Gly Ile Leu Pro Gly Cys Glu Pro Cys 1295 1300 1305His
Lys Lys Val Cys Ala His Gly Thr Cys Gln Pro Ser Ser Gln 1310 1315
1320Ala Gly Phe Thr Cys Glu Cys Gln Glu Gly Trp Met Gly Pro Leu
1325 1330 1335Cys Asp Gln Arg Thr Asn Asp Pro Cys Leu Gly Asn Lys
Cys Val 1340 1345 1350His Gly Thr Cys Leu Pro Ile Asn Ala Phe Ser
Tyr Ser Cys Lys 1355 1360 1365Cys Leu Glu Gly His Gly Gly Val Leu
Cys Asp Glu Glu Glu Asp 1370 1375 1380Leu Phe Asn Pro Cys Gln Ala
Ile Lys Cys Lys His Gly Lys Cys 1385 1390 1395Arg Leu Ser Gly Leu
Gly Gln Pro Tyr Cys Glu Cys Ser Ser Gly 1400 1405 1410Tyr Thr Gly
Asp Ser Cys Asp Arg Glu Ile Ser Cys Arg Gly Glu 1415 1420 1425Arg
Ile Arg Asp Tyr Tyr Gln Lys Gln Gln Gly Tyr Ala Ala Cys 1430 1435
1440Gln Thr Thr Lys Lys Val Ser Arg Leu Glu Cys Arg Gly Gly Cys
1445 1450 1455Ala Gly Gly Gln Cys Cys Gly Pro Leu Arg Ser Lys Arg
Arg Lys 1460 1465 1470Tyr Ser Phe Glu Cys Thr Asp Gly Ser Ser Phe
Val Asp Glu Val 1475 1480 1485Glu Lys Val Val Lys Cys Gly Cys Thr
Arg Cys Val Ser 1490 1495 15007240PRTHomo sapiens 7Ile Leu Asn Lys
Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys1 5 10 15Ser Gly Ser
Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro 20 25 30Arg Asn
Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn 35 40 45Ile
Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg 50 55
60Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala65
70 75 80Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn
His 85 90 95Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys
Leu Tyr 100 105 110Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile
Pro Arg Lys Ala 115 120 125Phe Arg Gly Ala Val Asp Ile Lys Asn Leu
Gln Leu Asp Tyr Asn Gln 130 135 140Ile Ser Cys Ile Glu Asp Gly Ala
Phe Arg Ala Leu Arg Asp Leu Glu145 150 155 160Val Leu Thr Leu Asn
Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser 165 170 175Phe Asn His
Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn 180 185 190Leu
Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln 195 200
205Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu
210 215 220Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val
Cys Ser225 230 235 2408465PRTHomo sapiens 8Ile Leu Asn Lys Val Ala
Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys1 5 10 15Ser Gly Ser Thr Val
Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro 20 25 30Arg Asn Ile Pro
Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn 35 40 45Ile Thr Arg
Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg 50 55 60Val Leu
Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala65 70 75
80Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His
85 90 95Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu
Tyr 100 105 110Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro
Arg Lys Ala 115 120 125Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln
Leu Asp Tyr Asn Gln 130 135 140Ile Ser Cys Ile Glu Asp Gly Ala Phe
Arg Ala Leu Arg Asp Leu Glu145 150
155 160Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala
Ser 165 170 175Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His
Ser Asn Asn 180 185 190Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser
Asp Trp Leu Arg Gln 195 200 205Arg Pro Arg Val Gly Leu Tyr Thr Gln
Cys Met Gly Pro Ser His Leu 210 215 220Arg Gly His Asn Val Ala Glu
Val Gln Lys Arg Glu Phe Val Cys Ser225 230 235 240Asp Glu Glu Glu
Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val 245 250 255Leu His
Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys 260 265
270Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile
275 280 285Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro
Pro Gly 290 295 300Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp
Leu Ser Asn Asn305 310 315 320Gln Ile Ser Glu Leu Ala Pro Asp Ala
Phe Gln Gly Leu Arg Ser Leu 325 330 335Asn Ser Leu Val Leu Tyr Gly
Asn Lys Ile Thr Glu Leu Pro Lys Ser 340 345 350Leu Phe Glu Gly Leu
Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn 355 360 365Lys Ile Asn
Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu 370 375 380Asn
Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly385 390
395 400Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln
Asn 405 410 415Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp
Tyr Leu His 420 425 430Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys
Thr Ser Pro Arg Arg 435 440 445Leu Ala Asn Lys Arg Ile Gly Gln Ile
Lys Ser Lys Lys Phe Arg Cys 450 455 460Ser4659692PRTHomo sapiens
9Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys1 5
10 15Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val
Pro 20 25 30Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly
Asn Asn 35 40 45Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg
His Leu Arg 50 55 60Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile
Glu Arg Gly Ala65 70 75 80Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu
Arg Leu Asn Arg Asn His 85 90 95Leu Gln Leu Phe Pro Glu Leu Leu Phe
Leu Gly Thr Ala Lys Leu Tyr 100 105 110Arg Leu Asp Leu Ser Glu Asn
Gln Ile Gln Ala Ile Pro Arg Lys Ala 115 120 125Phe Arg Gly Ala Val
Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln 130 135 140Ile Ser Cys
Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu145 150 155
160Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser
165 170 175Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser
Asn Asn 180 185 190Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp
Trp Leu Arg Gln 195 200 205Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys
Met Gly Pro Ser His Leu 210 215 220Arg Gly His Asn Val Ala Glu Val
Gln Lys Arg Glu Phe Val Cys Ser225 230 235 240Asp Glu Glu Glu Gly
His Gln Ser Phe Met Ala Pro Ser Cys Ser Val 245 250 255Leu His Cys
Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys 260 265 270Arg
Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile 275 280
285Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly
290 295 300Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser
Asn Asn305 310 315 320Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln
Gly Leu Arg Ser Leu 325 330 335Asn Ser Leu Val Leu Tyr Gly Asn Lys
Ile Thr Glu Leu Pro Lys Ser 340 345 350Leu Phe Glu Gly Leu Phe Ser
Leu Gln Leu Leu Leu Leu Asn Ala Asn 355 360 365Lys Ile Asn Cys Leu
Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu 370 375 380Asn Leu Leu
Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly385 390 395
400Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn
405 410 415Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr
Leu His 420 425 430Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr
Ser Pro Arg Arg 435 440 445Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys
Ser Lys Lys Phe Arg Cys 450 455 460Ser Gly Thr Glu Asp Tyr Arg Ser
Lys Leu Ser Gly Asp Cys Phe Ala465 470 475 480Asp Leu Ala Cys Pro
Glu Lys Cys Arg Cys Glu Gly Thr Thr Val Asp 485 490 495Cys Ser Asn
Gln Lys Leu Asn Lys Ile Pro Glu His Ile Pro Gln Tyr 500 505 510Thr
Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr Val Leu Glu Ala 515 520
525Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys Ile Asn Phe Ser
530 535 540Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe Glu Gly
Ala Ser545 550 555 560Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg
Leu Glu Asn Val Gln 565 570 575His Lys Met Phe Lys Gly Leu Glu Ser
Leu Lys Thr Leu Met Leu Arg 580 585 590Ser Asn Arg Ile Thr Cys Val
Gly Asn Asp Ser Phe Ile Gly Leu Ser 595 600 605Ser Val Arg Leu Leu
Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala 610 615 620Pro Gly Ala
Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn Leu Leu625 630 635
640Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp Leu Gly Glu Trp
645 650 655Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg Cys Gln
Lys Pro 660 665 670Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala
Ile Gln Asp Phe 675 680 685Thr Cys Asp Asp 69010886PRTHomo sapiens
10Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys1
5 10 15Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val
Pro 20 25 30Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly
Asn Asn 35 40 45Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg
His Leu Arg 50 55 60Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile
Glu Arg Gly Ala65 70 75 80Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu
Arg Leu Asn Arg Asn His 85 90 95Leu Gln Leu Phe Pro Glu Leu Leu Phe
Leu Gly Thr Ala Lys Leu Tyr 100 105 110Arg Leu Asp Leu Ser Glu Asn
Gln Ile Gln Ala Ile Pro Arg Lys Ala 115 120 125Phe Arg Gly Ala Val
Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln 130 135 140Ile Ser Cys
Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu145 150 155
160Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser
165 170 175Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser
Asn Asn 180 185 190Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp
Trp Leu Arg Gln 195 200 205Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys
Met Gly Pro Ser His Leu 210 215 220Arg Gly His Asn Val Ala Glu Val
Gln Lys Arg Glu Phe Val Cys Ser225 230 235 240Asp Glu Glu Glu Gly
His Gln Ser Phe Met Ala Pro Ser Cys Ser Val 245 250 255Leu His Cys
Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys 260 265 270Arg
Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile 275 280
285Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly
290 295 300Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser
Asn Asn305 310 315 320Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln
Gly Leu Arg Ser Leu 325 330 335Asn Ser Leu Val Leu Tyr Gly Asn Lys
Ile Thr Glu Leu Pro Lys Ser 340 345 350Leu Phe Glu Gly Leu Phe Ser
Leu Gln Leu Leu Leu Leu Asn Ala Asn 355 360 365Lys Ile Asn Cys Leu
Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu 370 375 380Asn Leu Leu
Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly385 390 395
400Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn
405 410 415Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr
Leu His 420 425 430Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr
Ser Pro Arg Arg 435 440 445Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys
Ser Lys Lys Phe Arg Cys 450 455 460Ser Gly Thr Glu Asp Tyr Arg Ser
Lys Leu Ser Gly Asp Cys Phe Ala465 470 475 480Asp Leu Ala Cys Pro
Glu Lys Cys Arg Cys Glu Gly Thr Thr Val Asp 485 490 495Cys Ser Asn
Gln Lys Leu Asn Lys Ile Pro Glu His Ile Pro Gln Tyr 500 505 510Thr
Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr Val Leu Glu Ala 515 520
525Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys Ile Asn Phe Ser
530 535 540Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe Glu Gly
Ala Ser545 550 555 560Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg
Leu Glu Asn Val Gln 565 570 575His Lys Met Phe Lys Gly Leu Glu Ser
Leu Lys Thr Leu Met Leu Arg 580 585 590Ser Asn Arg Ile Thr Cys Val
Gly Asn Asp Ser Phe Ile Gly Leu Ser 595 600 605Ser Val Arg Leu Leu
Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala 610 615 620Pro Gly Ala
Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn Leu Leu625 630 635
640Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp Leu Gly Glu Trp
645 650 655Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg Cys Gln
Lys Pro 660 665 670Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala
Ile Gln Asp Phe 675 680 685Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser
Cys Ser Pro Leu Ser Arg 690 695 700Cys Pro Thr Glu Cys Thr Cys Leu
Asp Thr Val Val Arg Cys Ser Asn705 710 715 720Lys Gly Leu Lys Val
Leu Pro Lys Gly Ile Pro Arg Asp Val Thr Glu 725 730 735Leu Tyr Leu
Asp Gly Asn Gln Phe Thr Leu Val Pro Lys Glu Leu Ser 740 745 750Asn
Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn Asn Arg Ile Ser 755 760
765Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln Leu Leu Thr Leu
770 775 780Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro Arg Thr
Phe Asp785 790 795 800Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His
Gly Asn Asp Ile Ser 805 810 815Val Val Pro Glu Gly Ala Phe Asn Asp
Leu Ser Ala Leu Ser His Leu 820 825 830Ala Ile Gly Ala Asn Pro Leu
Tyr Cys Asp Cys Asn Met Gln Trp Leu 835 840 845Ser Asp Trp Val Lys
Ser Glu Tyr Lys Glu Pro Gly Ile Ala Arg Cys 850 855 860Ala Gly Pro
Gly Glu Met Ala Asp Lys Leu Leu Leu Thr Thr Pro Ser865 870 875
880Lys Lys Phe Thr Cys Gln 885111092PRTHomo sapiens 11Ile Leu Asn
Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys1 5 10 15Ser Gly
Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro 20 25 30Arg
Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn 35 40
45Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg
50 55 60Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly
Ala65 70 75 80Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn
Arg Asn His 85 90 95Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr
Ala Lys Leu Tyr 100 105 110Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln
Ala Ile Pro Arg Lys Ala 115 120 125Phe Arg Gly Ala Val Asp Ile Lys
Asn Leu Gln Leu Asp Tyr Asn Gln 130 135 140Ile Ser Cys Ile Glu Asp
Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu145 150 155 160Val Leu Thr
Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser 165 170 175Phe
Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn 180 185
190Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln
195 200 205Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser
His Leu 210 215 220Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu
Phe Val Cys Ser225 230 235 240Asp Glu Glu Glu Gly His Gln Ser Phe
Met Ala Pro Ser Cys Ser Val 245 250 255Leu His Cys Pro Ala Ala Cys
Thr Cys Ser Asn Asn Ile Val Asp Cys 260 265 270Arg Gly Lys Gly Leu
Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile 275 280 285Thr Glu Ile
Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly 290 295 300Ala
Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn305 310
315 320Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser
Leu 325 330 335Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu
Pro Lys Ser 340 345 350Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu
Leu Leu Asn Ala Asn 355 360 365Lys Ile Asn Cys Leu Arg Val Asp Ala
Phe Gln Asp Leu His Asn Leu 370 375 380Asn Leu Leu Ser Leu Tyr Asp
Asn Lys Leu Gln Thr Ile Ala Lys Gly385 390 395 400Thr Phe Ser Pro
Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn 405 410 415Pro Phe
Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His 420 425
430Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg
435 440 445Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe
Arg Cys 450 455 460Ser Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly
Asp Cys Phe Ala465 470 475 480Asp Leu Ala Cys Pro Glu Lys Cys Arg
Cys Glu Gly Thr Thr Val Asp 485 490 495Cys Ser Asn Gln Lys Leu Asn
Lys Ile Pro Glu His Ile Pro Gln Tyr 500 505 510Thr Ala Glu Leu Arg
Leu Asn Asn Asn Glu Phe Thr Val Leu Glu Ala 515 520 525Thr Gly Ile
Phe Lys Lys Leu Pro Gln Leu Arg Lys Ile Asn Phe Ser 530 535 540Asn
Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe Glu Gly Ala Ser545 550
555 560Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu Glu Asn Val
Gln 565 570 575His Lys Met Phe
Lys Gly Leu Glu Ser Leu Lys Thr Leu Met Leu Arg 580 585 590Ser Asn
Arg Ile Thr Cys Val Gly Asn Asp Ser Phe Ile Gly Leu Ser 595 600
605Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala
610 615 620Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn
Leu Leu625 630 635 640Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala
Trp Leu Gly Glu Trp 645 650 655Leu Arg Lys Lys Arg Ile Val Thr Gly
Asn Pro Arg Cys Gln Lys Pro 660 665 670Tyr Phe Leu Lys Glu Ile Pro
Ile Gln Asp Val Ala Ile Gln Asp Phe 675 680 685Thr Cys Asp Asp Gly
Asn Asp Asp Asn Ser Cys Ser Pro Leu Ser Arg 690 695 700Cys Pro Thr
Glu Cys Thr Cys Leu Asp Thr Val Val Arg Cys Ser Asn705 710 715
720Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg Asp Val Thr Glu
725 730 735Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro Lys Glu
Leu Ser 740 745 750Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn
Asn Arg Ile Ser 755 760 765Thr Leu Ser Asn Gln Ser Phe Ser Asn Met
Thr Gln Leu Leu Thr Leu 770 775 780Ile Leu Ser Tyr Asn Arg Leu Arg
Cys Ile Pro Pro Arg Thr Phe Asp785 790 795 800Gly Leu Lys Ser Leu
Arg Leu Leu Ser Leu His Gly Asn Asp Ile Ser 805 810 815Val Val Pro
Glu Gly Ala Phe Asn Asp Leu Ser Ala Leu Ser His Leu 820 825 830Ala
Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn Met Gln Trp Leu 835 840
845Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly Ile Ala Arg Cys
850 855 860Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu Thr Thr
Pro Ser865 870 875 880Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val
Asn Ile Leu Ala Lys 885 890 895Cys Asn Pro Cys Leu Ser Asn Pro Cys
Lys Asn Asp Gly Thr Cys Asn 900 905 910Ser Asp Pro Val Asp Phe Tyr
Arg Cys Thr Cys Pro Tyr Gly Phe Lys 915 920 925Gly Gln Asp Cys Asp
Val Pro Ile His Ala Cys Ile Ser Asn Pro Cys 930 935 940Lys His Gly
Gly Thr Cys His Leu Lys Glu Gly Glu Glu Asp Gly Phe945 950 955
960Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn Cys Glu Val Asn
965 970 975Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn Ser Thr
Cys Val 980 985 990Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro
Glu Tyr Thr Gly 995 1000 1005Glu Leu Cys Glu Glu Lys Leu Asp Phe
Cys Ala Gln Asp Leu Asn 1010 1015 1020Pro Cys Gln His Asp Ser Lys
Cys Ile Leu Thr Pro Lys Gly Phe 1025 1030 1035Lys Cys Asp Cys Thr
Pro Gly Tyr Val Gly Glu His Cys Asp Ile 1040 1045 1050Asp Phe Asp
Asp Cys Gln Asp Asn Lys Cys Lys Asn Gly Ala His 1055 1060 1065Cys
Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile Cys Pro Glu Gly 1070 1075
1080Tyr Ser Gly Leu Phe Cys Glu Phe Ser 1085 1090121165PRTHomo
sapiens 12Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys
Ser Cys1 5 10 15Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg
Ser Val Pro 20 25 30Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu
Asn Gly Asn Asn 35 40 45Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly
Leu Arg His Leu Arg 50 55 60Val Leu Gln Leu Met Glu Asn Lys Ile Ser
Thr Ile Glu Arg Gly Ala65 70 75 80Phe Gln Asp Leu Lys Glu Leu Glu
Arg Leu Arg Leu Asn Arg Asn His 85 90 95Leu Gln Leu Phe Pro Glu Leu
Leu Phe Leu Gly Thr Ala Lys Leu Tyr 100 105 110Arg Leu Asp Leu Ser
Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala 115 120 125Phe Arg Gly
Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln 130 135 140Ile
Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu145 150
155 160Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala
Ser 165 170 175Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His
Ser Asn Asn 180 185 190Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser
Asp Trp Leu Arg Gln 195 200 205Arg Pro Arg Val Gly Leu Tyr Thr Gln
Cys Met Gly Pro Ser His Leu 210 215 220Arg Gly His Asn Val Ala Glu
Val Gln Lys Arg Glu Phe Val Cys Ser225 230 235 240Asp Glu Glu Glu
Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val 245 250 255Leu His
Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys 260 265
270Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile
275 280 285Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro
Pro Gly 290 295 300Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp
Leu Ser Asn Asn305 310 315 320Gln Ile Ser Glu Leu Ala Pro Asp Ala
Phe Gln Gly Leu Arg Ser Leu 325 330 335Asn Ser Leu Val Leu Tyr Gly
Asn Lys Ile Thr Glu Leu Pro Lys Ser 340 345 350Leu Phe Glu Gly Leu
Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn 355 360 365Lys Ile Asn
Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu 370 375 380Asn
Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly385 390
395 400Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln
Asn 405 410 415Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp
Tyr Leu His 420 425 430Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys
Thr Ser Pro Arg Arg 435 440 445Leu Ala Asn Lys Arg Ile Gly Gln Ile
Lys Ser Lys Lys Phe Arg Cys 450 455 460Ser Gly Thr Glu Asp Tyr Arg
Ser Lys Leu Ser Gly Asp Cys Phe Ala465 470 475 480Asp Leu Ala Cys
Pro Glu Lys Cys Arg Cys Glu Gly Thr Thr Val Asp 485 490 495Cys Ser
Asn Gln Lys Leu Asn Lys Ile Pro Glu His Ile Pro Gln Tyr 500 505
510Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr Val Leu Glu Ala
515 520 525Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys Ile Asn
Phe Ser 530 535 540Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe
Glu Gly Ala Ser545 550 555 560Gly Val Asn Glu Ile Leu Leu Thr Ser
Asn Arg Leu Glu Asn Val Gln 565 570 575His Lys Met Phe Lys Gly Leu
Glu Ser Leu Lys Thr Leu Met Leu Arg 580 585 590Ser Asn Arg Ile Thr
Cys Val Gly Asn Asp Ser Phe Ile Gly Leu Ser 595 600 605Ser Val Arg
Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala 610 615 620Pro
Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn Leu Leu625 630
635 640Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp Leu Gly Glu
Trp 645 650 655Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg Cys
Gln Lys Pro 660 665 670Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val
Ala Ile Gln Asp Phe 675 680 685Thr Cys Asp Asp Gly Asn Asp Asp Asn
Ser Cys Ser Pro Leu Ser Arg 690 695 700Cys Pro Thr Glu Cys Thr Cys
Leu Asp Thr Val Val Arg Cys Ser Asn705 710 715 720Lys Gly Leu Lys
Val Leu Pro Lys Gly Ile Pro Arg Asp Val Thr Glu 725 730 735Leu Tyr
Leu Asp Gly Asn Gln Phe Thr Leu Val Pro Lys Glu Leu Ser 740 745
750Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn Asn Arg Ile Ser
755 760 765Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln Leu Leu
Thr Leu 770 775 780Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro
Arg Thr Phe Asp785 790 795 800Gly Leu Lys Ser Leu Arg Leu Leu Ser
Leu His Gly Asn Asp Ile Ser 805 810 815Val Val Pro Glu Gly Ala Phe
Asn Asp Leu Ser Ala Leu Ser His Leu 820 825 830Ala Ile Gly Ala Asn
Pro Leu Tyr Cys Asp Cys Asn Met Gln Trp Leu 835 840 845Ser Asp Trp
Val Lys Ser Glu Tyr Lys Glu Pro Gly Ile Ala Arg Cys 850 855 860Ala
Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu Thr Thr Pro Ser865 870
875 880Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val Asn Ile Leu Ala
Lys 885 890 895Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn Asp Gly
Thr Cys Asn 900 905 910Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr Cys
Pro Tyr Gly Phe Lys 915 920 925Gly Gln Asp Cys Asp Val Pro Ile His
Ala Cys Ile Ser Asn Pro Cys 930 935 940Lys His Gly Gly Thr Cys His
Leu Lys Glu Gly Glu Glu Asp Gly Phe945 950 955 960Trp Cys Ile Cys
Ala Asp Gly Phe Glu Gly Glu Asn Cys Glu Val Asn 965 970 975Val Asp
Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn Ser Thr Cys Val 980 985
990Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro Glu Tyr Thr Gly
995 1000 1005Glu Leu Cys Glu Glu Lys Leu Asp Phe Cys Ala Gln Asp
Leu Asn 1010 1015 1020Pro Cys Gln His Asp Ser Lys Cys Ile Leu Thr
Pro Lys Gly Phe 1025 1030 1035Lys Cys Asp Cys Thr Pro Gly Tyr Val
Gly Glu His Cys Asp Ile 1040 1045 1050Asp Phe Asp Asp Cys Gln Asp
Asn Lys Cys Lys Asn Gly Ala His 1055 1060 1065Cys Thr Asp Ala Val
Asn Gly Tyr Thr Cys Ile Cys Pro Glu Gly 1070 1075 1080Tyr Ser Gly
Leu Phe Cys Glu Phe Ser Pro Pro Met Val Leu Pro 1085 1090 1095Arg
Thr Ser Pro Cys Asp Asn Phe Asp Cys Gln Asn Gly Ala Gln 1100 1105
1110Cys Ile Val Arg Ile Asn Glu Pro Ile Cys Gln Cys Leu Pro Gly
1115 1120 1125Tyr Gln Gly Glu Lys Cys Glu Lys Leu Val Ser Val Asn
Phe Ile 1130 1135 1140Asn Lys Glu Ser Tyr Leu Gln Ile Pro Ser Ala
Lys Val Arg Pro 1145 1150 1155Gln Thr Asn Ile Thr Leu Gln 1160
1165
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